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Immunohistochemistry, and
Antigen Retrieval Methods
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Microscopy,
Immunohistochemistry, and
Antigen Retrieval Methods
For Light and Electron Microscopy
M. A. Hayat
Kean University
Union, New Jersey
No part of this eBook may be reproduced or transmitted in any form or by any means, electronic,
mechanical, recording, or otherwise, without written consent from the Publisher
There are several important reasons for publishing this book. One reason is to present
chemical and physical principles governing the processing of tissues using microwave
heating as an adjunct to fixation, embedding in a resin, and staining. A second reason is to
point interested readers to a number of recent developments in the retrieval and localiza-
tion of antigens in normal and pathological tissues. The greatest concentration of work in
this field has focused on the detectability of disease-related proteins. Therefore, as exam-
ples, the detectability and the role in disease of estrogens, p53, p185, Ki-67, and PCNA are
discussed in detail.
A third reason is to review favorable aspects of the histochemical approach, whereby
it yields data not obtainable by any other means, including biochemical assays.
Histochemistry, for instance, contributes to acquiring knowledge about the biological activ-
ity of normal and diseased cells, which is supported by illustrations. Immunohistochemistry
defines the function of cell types in a tissue and organs by localizing and identifying their
contents or products. This methodology is highly visual; illustrations, especially color
images, often contribute as much to correct understanding and interpretation of the results
as the text. Therefore, the results of many methods are illustrated.
During the last decade there has been significant progress in understanding the
mechanisms responsible for antigen masking during fixation and subsequent unmasking,
primarily by heating or, in some cases, by enzymatic digestion. Comparative studies
demonstrate, for example, that not only microwave heating but also other sources of
heating are effective in antigen retrieval. Similar studies also indicate that although
sodium citrate buffer is in common use as the antigen retrieval fluid, unmasking of certain
antigens requires other fluids. These and other new developments are discussed in this
volume.
In preparing the reader to study the location of proteins and carbohydrates, it is nec-
essary to explain the advantages and limitations of the study. A potential limitation of the
immunohistochemical approach arises from the possibility of false-negative staining due
to the failure of an antibody to yield positive results. It is equally important to be aware of
the possibility of false-positive staining, which can arise if the method is not scrupulous
regarding histochemical negative and positive controls. Suggestions are offered to at least
minimize these histological artifacts. In this regard the importance of negative and positive
controls cannot be overemphasized. Negative controls involve the omission of the primary
vii
viii Preface
The help and encouragement received from Dean Betty Barber throughout the writ-
ing of this book are greatly appreciated and will be remembered. I thank Patricia Lemus
and Elizabeth McGovern for their expert secretarial assistance in the preparation of the
manuscript, and I appreciate the help and cooperation extended to me by Roberta Klarreich,
the production editor, throughout the production of this volume.
M. A. Hayat
October 2001
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Contents
Chapter 1. Introduction 1
xi
xii Contents
Antibody Cross-Reactivity 48
Polyreactive Antibodies 49
Commercial Sources of Antibodies 50
Formaldehyde 53
Nature of Formaldehyde Solution 54
Mechanism of Fixation with Formaldehyde 54
Comparison of Formaldehyde with Glutaraldehyde 56
Fixation with Formaldehyde 57
Effect of Prolonged Fixation with Formaldehyde 58
Formalin Substitute Fixatives 59
Fixation Conditions 60
Effect of Heating on Fixation with Glutaraldehyde 61
Microwave Heat–Assisted Fixation with Osmium Tetroxide 62
Role of Microwave Heating in Enzyme Cytochemistry 64
Fixation for Enzyme Cytochemistry Using Microwave at Relatively
Low Temperature 64
Cryopreservation in the Presence of Microwave Heating 65
Paraffin Embedding 65
Paraffin Embedding in Microwave Oven 67
Paraffin Embedding in Vacuum-Microwave Oven 67
Microtomy of Paraffin-Embedded Tissues 67
Silanting of Glass Slides 68
Vacuum-Assisted Microwave Heating 69
Procedure 181
Microwave Heat–Assisted Enhanced Peroxidase
One-Step Method 181
Procedure 181
Microwave Heat–Assisted Immunostaining of Cell Smears 182
Double Immunostaining Using Microwave Heating 182
Microwave Heat–Enhanced Double Immunostaining of Nuclear
and Cytoplasmic Antigens 183
Procedure 183
Microwave Heat–Assisted Immunohistochemical Localization of
Cyclin D1 184
Microwave Heat–Assisted Immunofluorescence Staining of Tissue
Sections 185
Procedure 186
Microwave Heat–Assisted Double Immunofluorescence Labeling 186
Procedure 186
Microwave Heat–Assisted Double Indirect Immunofluorescence
Staining 187
Procedure 187
Control Procedures 188
Immunoenzymatic Detection 189
Combined Microwave Heating and Ultrasound Antigen
Retrieval Method 189
Combined Enzyme Digestion and Microwave Heating
Antigen Retrieval Method 190
Pressure Cooker–EDTA–Assisted Antigen Retrieval 191
2-Mercaptoethanol–Sodium Iodoacetate–Assisted
Antigen Retrieval 191
Antigen Retrieval with Steam–EDTA–Protease Method 192
Procedure 192
Picric Acid–Steam Autoclaving–Formic Acid–Guanidine
Thiocyanate–Assisted Retrieval of Prion Protein 192
Procedure 194
Simultaneous Detection of Multiple Antigens 194
Procedure 196
Use of Multiple Antibodies for Labeling Antigens 196
Procedure 197
Antigen Retrieval in Neuronal Tissue Slices before Vibratome
Sectioning 198
Microwave Heat–Assisted Antigen Retrieval in Freshly Frozen
Brain Tissue 198
Procedure 199
Microwave Heat–Assisted Rapid Immunostaining of Frozen
Sections 199
Procedure 200
Microwave Heat–Assisted Immunocytochemistry of Thin
Cryosections 200
Procedure 201
xvi Contents
References 305
Index 351
Microscopy,
Immunohistochemistry, and
Antigen Retrieval Methods
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Chapter 1
Introduction
1
2 Chapter 1
The other point of view, which favors the term antigen retrieval instead of epitope
retrieval, is as follows (S.-R. Shi, personal communication). It is likely that the mechanism
of antigen retrieval is based on chemical modification of protein conformation. Therefore,
retrieval of formalin-modified (or masked) antigenicity must be a restoration of the protein
structure, as any antigen/antibody recognition is dependent on protein conformation. This
is particularly true for discontinuous epitopes (most antigen determinants are discontinu-
ous epitopes), which consist of amino acid sequences apart from each other on one
polypeptide (or actually located on distinct polypeptides) but brought near each other in
the tertiary or quaternary structure of the protein. In other words, restoration of the func-
tion of an epitope (antigenicity) is the retrieval of its protein conformation, i.e., retrieval of
antigen. Because the concept of epitope is not an intrinsic feature of a protein existing
independently of its paratope partner, the term epitope refers to only a functional unit, but
not a stoic structure of the protein. Shi et al. (2000a) have further justified the use of the
term antigen and rejected the relevancy of the term epitope in immunohistochemistry.
In support of Dr. Shi’s opinion is the fact that in some cases the absolute specificity
of even monoclonal antibodies can be questioned. The absorption control cannot always
determine whether the protein bound in the tissue is the same protein used for absorption.
The monoclonal antibody may instead recognize a similar epitope of an unrelated protein,
especially following tissue fixation. Absorption controls therefore may not provide the
specificity of the antibody for a protein under study in the tissue.
In light of the above-mentioned difference of opinion, and in the absence of a definite
understanding regarding unmasking of an epitope or whole antigen molecule as a result of
unmasking treatments (heat or nonheat treatment), both terms, epitopes and antigens, are
used in this volume.
Immunohistochemistry has surpassed other techniques in its effectiveness in the
in situ preservation and detection of antigens. Immunopathology has become a valuable or
even an essential adjunct to diagnostic pathology. It is affirmed that diagnostic immuno-
histochemistry is indispensable in surgical pathology for diagnosis, therapy, and progno-
sis. The usefulness of this methodology depends and will continue to rely on three major
factors: (1) availability of specific primary antibodies; (2) an efficient detection system;
and (3) correct interpretation and significance of the findings. An increasing number of
monoclonal antibodies is being produced, and many of them are commercially available;
these sources are given in Chapter 2. The role of antibodies in diagnostic pathology is dis-
cussed in this chapter. Highly sensitive detection systems are available, and their signals
are being continuously enhanced to achieve high signal-to-noise ratio. Methods of scoring
the signals are also available. These improvements are discussed in Chapter 4.
All immunohistological methods depend on the successful completion of a series of
sequential steps, beginning with specimen collection; their morphological and antigenic
preservation (chemical fixation or rapid freezing) or antigenic retrieval; incubation in an
antibody or sequence of antibodies; staining; signal counting; and interpretation of results.
Each of these steps must be performed as efficiently and correctly as possible. If even one
of these steps is suboptimal, the remaining steps, even though perfectly carried out, can
never compensate for the inefficient step. Any error in immunohistochemistry, especially
when applied to clinical diagnosis, is unacceptable. This methodology is not only a science
but also an art; each aspect depends on the other.
Progress in molecular biology is intimately related to advancement in technology.
One fundamental goal of cell research is to understand the functions of molecules that
Introduction 3
constitute cells and tissues. This understanding can be enhanced by examining the molec-
ular details and subcellular location of cell components. The precise extracellular and
intracellular localization of molecules under different physiological and pathological con-
ditions yields clues to their possible functions. These aspects of antigen molecules and
receptors, especially clinically important ones such as p53, Ki-67, PCNA, p185, and estro-
gens, are discussed in detail in Chapters 10, 11, and 12.
The achievement of the above-mentioned goal received an impetus from the devel-
opment of the heating methodology (especially microwave heating) for antigen retrieval.
Microwave heating was introduced into biomedical research approximately two decades
ago for the rapid processing of plant and animal tissues. The development of this technique
was a significant step forward in the application of histochemistry, immunohistochemistry,
and immunocytochemistry. In other words, this methodology has significantly contributed
to the localization of macromolecules and molecules (including antigens) and thus to an
understanding of their functions. This technique is also useful for enhancing the detection
of RNA and DNA by in situ hybridization (see page 213). Another example of the appli-
cation of microwave heating is in conjunction with flow cytometry (see page 225). The
polymerase chain reaction (PCR) has also been used in conjunction with microwave
heating for studying DNA (see page 224). Yet another application of microwave heating is
with the enzyme-linked-immunosorbent assay method (ELISA) (see page 228). Tissue
cryopreservation with diminished ice crystal growth has also been accomplished with this
versatile technology (see page 65). Application of microwave heating to enzyme cyto-
chemistry, autoradiography, and X-ray microanalysis has been attempted (Mizuhira and
Hasegawa, 1996).
Significant aspects of the basic biology of disease processes are now assuming clini-
cal importance in diagnosis and prognosis. The pathologist can identify an ever-increasing
range of antigens in tissue sections using the techniques mentioned above. Identification
of tissue antigens using these methods is of fundamental importance for clarifying tumor
proteins or carbohydrates, determining the diagnosis and prognosis of tumors, character-
izing pervasive nepotistic alterations in tissues such as prostate, subclassifying neoplasms,
evaluating the response of tumors and pervasive nepotistic changes to certain therapies
(i.e., as a surrogate intermediate and end point), selecting patients who are candidates for
specific therapies (e.g., immunotherapy), and identifying pathogenic organisms (Arnold
et al., 1996). For these and other reasons, immunohistochemistry has become the most
important tool in research and diagnostic pathology. It permits detection of defined anti-
gens on cryostat, paraffin, and resin sections of normal and diseased tissues.
Immunohistochemical and immunocytochemical localization of antigens is a power-
ful tool that provides insight into some of the salient features of cell and tissue complex-
ity. Such studies, for example, demonstrate relationships between normal cell structure and
function and pathological consequences. Presently, immunohistochemistry is firmly estab-
lished as the most important method for detecting antigens with the light microscope. It
can be effectively used to examine various antigens in the sections of formaldehyde-fixed
and paraffin-embedded tissues (Hayat, 2000a). The availability of the equipment to carry
out immunohistochemistry and the introduction of many new monoclonal antibodies make
it possible to apply this technique to retrospective studies.
The introduction of a large number of new monoclonal antibodies of improved sensi-
tivity and specificity, which are available in ready-to-use kits, has made possible a wider
use of immunohistochemistry for antigen analysis. In addition, the development of various
4 Chapter 1
antigen retrieval methods during the past two decades has enabled many more antibodies
to access antigens that were undetectable or minimally detectable in the past. Today almost
any antigen that survives tissue processing has the potential to be localized immunohisto-
chemically. As a result of these methods, additional antibodies have become paraffin- and
resin-compatible, which permits heat-treated tissue sections to be used for detecting anti-
gens with the light and electron microscopes.
New antigen retrieval methods, especially microwave heating and other heating pro-
cedures and ultrasound treatment, can effectively retrieve antigens from tissues left in
formaldehyde for prolonged periods. The introduction of the computer-assisted image ana-
lyzer and other automated equipment (e.g., the automatic stainer), and generation of anti-
bodies to synthetic peptides, have ushered immunohistochemistry into a higher level of
efficiency, accuracy, and quantitation. The demand for a more precise spatial localization
of epitopes favors the use of antibody fragments (e.g., Fab), peptides, or ligands.
These advances facilitate the use of antigen detection for correct diagnosis and prog-
nosis. Furthermore, advances in detection accuracy provide guidelines to study and
understand more complicated biological problems. To achieve these goals, standardization
of antigen retrieval methods is necessary, at the least, to minimize inter- and intralabora-
tory (including interobserver) variability of immunostaining (see Chapter 5). However,
even in the absence of such standardization, the method has become the most effective tool
in light microscope immunohistochemistry and, to some extent, in electron microscope
immunocytochemistry.
Introduction 5
Although antigen retrieval is carried out most commonly on paraffin sections, it can
also be accomplished on semithin or thin resin sections for light and electron microscopy,
respectively. Thin sections of routinely used resins such as epoxy, LR White, LR Gold,
and Lowicryls can be used for detecting antigens with the light or electron microscope.
These resins, in conjunction with microwave heating, can also be used for cell and ultra-
structural studies with the light microscope, and scanning and transmission electron
microscope (Fig. 1.1).
This procedure can also be employed for studying bacteria with the scanning elec-
tron microscope (Fig. 1.2).
The advantages of resin sections include better preservation of cellular details, assist-
ing the achievement of higher resolution, and the ability to carry out correlative studies of
the same tissue with the light microscope and scanning and transmission electron micro-
scope (Fig. 1.3). In addition, resin sections (also cryosections) allow immunogold and
silver-enhanced immunogold staining.
Biochemical assays such as the dextran-coated charcoal (DCC) assay, certain signal
amplification techniques, and other cytosol-based methods have been mostly replaced
6 Chapter 1
by immunohistochemistry because the former methods are costly and often difficult to
reproduce. For example, the DCC assay requires rather large tissue specimens and may be
adversely influenced by tissue heterogeneity (e.g., tumors), presence of bound endogenous
estrogen, and sampling error (Hendricks and Wilkinson, 1993). In cytosol-based biochem-
ical assays, tissues are indiscriminately homogenized (e.g., tumor, stroma, inflammatory
cells, and epithelial cells). Therefore, the results expressed in fentomoles per milligram of
the total protein are variably diluted because of the presence of nontumorous cells.
In contrast, immunohistochemistry can be carried out with smaller tissue specimens
and is less affected by tissue heterogeneity or endogenous hormones. This technique
allows direct histological visualization, which permits separation of tumors from stroma,
inflammatory cells, and normal cells. However, the DCC assay can be employed for cor-
relative studies; therefore, the selection of a particular method in a diagnostic laboratory
should depend on a number of factors, including specificity, sensitivity, rapidity, use of
potentially harmful reagents, availability of equipment, cost, and application to a wide
range of antigens.
However, the central problem in immunohistochemistry is to retain antigenicity with-
out sacrificing the quality of cell morphological preservation. It has been established that
the preservation of antigenicity is inversely related to the preservation of cell morphology
(Hayat, 2000a); thus, tissue preparation methods optimal for the preservation of cell mor-
phology introduce protein crosslinking and are therefore suboptimal for preserving anti-
genicity. Because preserving antigenicity in immunohistochemistry is more important than
preserving morphology, 10% formaldehyde is generally used for fixation. Glutaraldehyde,
on the other hand, yields excellent ultrastructural preservation but severely masks most
antigens by introducing irreversible protein crosslinking. The mechanism(s) responsible
Introduction 7
8 Chapter 1
for the antigen-masking effects of fixatives are discussed in Chapter 4. Tissue embedding
in paraffin also adversely affects antigen detection.
The alternative to chemical fixation and paraffin embedding is to use frozen sections
of fresh-frozen tissues (snap freezing). These cryostat sections tend to provide improved
antigen detection. Proteins are retained in these sections at least until the cryosections are
placed into aqueous incubation and staining solutions. Most antibodies recognize antigens
better in frozen sections than in sections of chemically fixed and paraffin-embedded tissues.
Fixation of frozen sections has also been recommended by some workers to immobilize
proteins during subsequent processing of cryostat sections and thus improve the antigen
localization. However, an agreement on the beneficial effect of this practice is lacking.
Moreover, cryostat sections are difficult to prepare, and the quality of morphological preser-
vation is comparatively poor (Fig. 1.4).
Fortunately, methods are available that satisfactorily preserve cell morphology as
well as antigenicity. The best compromise is to use a mixture of 6% formaldehyde and glu-
taraldehyde of a low concentration (0.1–0.4%). This approach, which is used extensively
for electron microscopy, should be used for light microscopy and is presented in this
volume. The optimal immunohistochemical method should ideally detect all specific epi-
topes, but in practice this is not possible. The best that can be accomplished is to optimize
the protocol to detect all the detectable epitopes.
Presently, the majority of the antigen retrieval studies are carried out using microwave
heating, although other heating methods such as autoclaving (page 145) or pressure cooking
(pages 127–128) can be equally effective, depending on the types of tissue and antigen
under study. Antigen retrieval can also be accomplished in a microwave oven under vac-
uum, as can rapid fixation, resin embedding, and staining. Ultrafast (milliseconds) or fast
(seconds to minutes) fixation can be achieved with this method. Even rapid fixation with
osmium tetroxide can be carried out in a microwave oven (see Chapter 3).
Like any other technique, microwave heating has certain limitations. A well-known
example is the uneven distribution of hot and cold spots in the oven, although this can be
minimized by using a water load in the oven. Prior to placing the water load in the oven,
cold and hot spots should be located in the oven cavity by using a high-brightness neon
bulb array. Hot spots must be avoided because an excessive increase in temperature dur-
ing irradiation is a major cause of poor fixation. The problem of hot spots apparently can
be avoided by using heating methods other than microwave heating. One should also be
aware that microwave heating may promote cross-reactivity by producing or unmasking
antigens closely related to those under study. The result may be the decreased specificity
of an established and generally available antibody (Alexander and Dayal, 1997). Other
limitations of microwave heating are listed on pages 142-144.
Antigen retrieval can also be accomplished by treating paraffin sections with diges-
tive enzymes. A number of proteolytic enzymes, including trypsin, pepsin, pronase, and
ficin (a plant enzyme), have been used for unmasking antigens. However, effectiveness of
each enzyme is limited to a few types of antigens. Moreover, enzymatic digestion is inef-
fective for antigen retrieved in overfixed tissues and tends to damage cell morphology,
especially when the treatment is prolonged. Cumulative evidence indicates that heating is
generally better than digestion. Therefore, the latter approach is not preferred unless the
former is unsuccessful. Better results are obtained in some cases when enzyme digestion
is used in conjunction with heat treatment.
Introduction 9
10 Chapter 1
Monoclonal antibody MIB-1 is used to recognize this antigen, and so the usefulness of this
antibody is discussed in detail in Chapter 2. Immunohistochemical methods using
microwave heating or autoclave treatment for localizing Ki-67 are presented.
Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase
8 and is intimately associated with DNA replication. Indeed, direct interaction between
DNA polymerase and its processivity factor PCNA is essential for effective replication of
the eukaryotic genome. This protein also plays a key role in other functions, such as
nucleotide excision repair, mismatch repair, base excision repair, cell cycle control, apop-
tosis, and transcription. These interactions support the concept that PCNA plays a central
role in connecting all these important cellular processes and can function as cellular com-
municator in cells. Clinically useful activity of PCNA can be identified by immunohisto-
chemistry and flow cytometry. Expression of this antigen in a cell population equates to
the growth fraction, that is, the proportion of cells involved in an active cell cycle. Because
PCNA is expressed in all cycling cells, the entire proportion of dividing cells present at any
instant in a population can be detected. Details of immunohistochemical staining of PCNA
on cryostat and paraffin-embedded sections are presented in this volume.
In recent years evidence has increasingly demonstrated the importance of proto-
oncogenes in the pathogenesis of diseases, including breast cancer. Amplification of
HER-2/neu gene is found in ~25% of human breast cancers and results in the overex-
pression of p185 oncoprotein. Amplification of the gene in breast cancer patients is corre-
lated with shorter disease-free states and poorer overall survival rates than in patients
showing no such amplification. Accurate detection of the gene amplification in breast can-
cer tissues is important in determining patient prognosis as well as response to standard
chemotherapeutic agents. Moreover, it is currently the sole criterion for selecting patients
for HER-2/neu-targeted therapy with the recombinant humanized anti-p185 antibody
Herceptin (trastuzumab) (Pauletti et al., 2000). Overexpression of this protein in breast
cancer is associated with adverse prognostic factors that include advanced pathological
stage, number of metastatic axillary lymph nodes, absence of estrogen and progesterone
receptors, increased S-phase fraction, DNA ploidy, and high nuclear grade.
Because the gene product is ultimately responsible for the biological activity of the
gene, it is apparent that direct measurement of the protein or immunohistochemical analysis
is as clinically relevant as is the determination of the number of gene copies (Battifora et al.,
1991). Immunohistochemistry is superior to biochemical assays because it eliminates the
dilution effect caused by variable amounts of stroma and other nonneoplastic tissues.
Another advantage of immunohistochemistry is its ability to detect overproduction of an
oncoprotein resulting from a mechanism other than gene amplification. Amplification of
HER-2/neu gene and Overexpression of p185 are also found in many tissues other than those
with breast cancer. Immunohistochemical detection of p185 is presented in Chapter 12.
sites of gene deregulation for molecular analysis, can provide an important adjunct to
diagnostic surgical pathology. For example, karyotypic analyses are helpful in the differ-
ential diagnosis of histologically similar small round cell tumors, including lymphoma and
neuroblastoma (Sreekantaiah et al., 1994). These tumors are composed of primitive cells
that often lack distinguishing features.
Each of these tumors contains specific chromosome changes; thus, cytogenetic analy-
sis provides a reliable approach that can distinguish between these neoplasms. In addition,
the identification of diagnostic chromosome translocations in histologically undifferenti-
ated tumors may support a diagnosis that is doubtful on histological grounds alone or may
even lead to a reconsideration of the histological diagnosis. This approach can aid in
directing therapy, determining prognosis, and identifying sites of gene perturbation for
molecular characterization.
Unfortunately, cytogenetic evaluation of tumors is still a relatively underutilized
approach. However, new potentially promising tumor markers have been introduced based
on the molecular genetic cancer research. Various genetic alterations important in carcino-
genesis, of which alterations in the ras oncogenes and the p53 tumor suppressor gene are the
most common, have now been described. Both of these are useful targets for diagnostic pur-
poses. This is substantiated by considering that p53 alterations are among the most frequent
genetic alterations in human malignancies. Similarly, clinical application of ras gene muta-
tion, for example in the diagnosis of pancreatic adenocarcinoma, has been well established
(e.g., Berthelemy et al., 1995). Chromosomal abnormalities in many tumors and their diag-
nostic relevance are discussed by Sreekantaiah et al. (1994) and Gisselsson et al. (2001).
Finally, cancer is a genetic disease, for acquired genetic aberrations cause the disease.
Changes in antigen expression detected with immunohistochemistry in some instances
reflects genetic alterations. The detection of these aberrations at the chromosome and gene
levels improved diagnosis, prognosis, and therapy. Therefore, the combination of morphol-
ogy with genetics is a major step toward a better understanding of human disease. A num-
ber of techniques that facilitate this combination are available, such as in situ hybridization,
comparative genomic hybridization, expression profiling using array technologies, high
throughput screening approaches, and phenotype/genotype correlations on the DNA,
RNA, or protein level (Ried et al., 1999). Technological innovations such as image analy-
sis systems, cytophotometric and integrated densitometric quantitation, and computer
hard- and software development also assist in this effort.
or gains of whole chromosomes or large portions of them (Lengauer et al., 1998). On the
other hand, in a small subset of tumors, genetic instability is observed at the nucleotide
level and results in base substitutions, deletions, or insertions of a few nucleotides. An
understanding of these instabilities is providing new insights into tumor pathogenesis.
Four major types of genetic alterations that affect growth-controlling genes have been
identified in neoplastic cells and are the basis of human cancers.
1. Sequence changes involving base substitutions, deletions, or insertions of a few
nucleotides. This type of subtle change is exemplified by missense mutations in
the K-ras gene, which occur in more than 80% of pancreatic cancers (Almoguera
et al., 1988). These changes cannot be detected with cytogenetic analysis.
2. Alterations in chromosome number involve losses or gains of whole chromosomes
and are found in almost all major types of human tumors. Losses of heterozygosity
(losses of a maternal or paternal allele) are widespread. The average cancer of the
colon, breast, pancreas, or prostate may lose ~25% of its alleles, and some tumors
may lose more than half of their alleles (e.g., Vogelstein et al., 1989). Such can-
cers exhibit a true chromosomal instability that persists throughout the lifetime of
the tumor. It is known that chromosome 10 is lost in glioblastomas, inactivating
the tumor suppressor gene PTEN (Wang et al., 1997). The gain of chromosome 7
in papillary renal carcinomas indicates a duplication of a mutant MET oncogene
(Zhuang et al., 1998).
3. Chromosome translocations are common in certain human cancers. Translocations
such as fusions of different chromosomes or of normally noncontiguous segments of
a single chromosome can be detected cytogenetically. At the molecular level, such
translocations can produce fusions between two different genes, imparting to the
fused transcript the tumorigenic properties. For example, in chronic myelogenous
leukemias the carboxy terminus of the c-abl gene on chromosome 9 is joined to the
amino terminus of the BCR gene on chromosome 22 (Nowell, 1997).
Translocation can also cause gains or losses of chromosomal material and
generate new gene products. Simple translocations are characterized by distinctive
rearrangements of chromosomal segments in specific neoplastic diseases, includ-
ing leukemias and lymphomas. These specific translocations are necessary for the
development and progression of the neoplasms in which they occur.
4. Gene amplification is an important process in human cancers, as it is associated
with tumor progression, has prognostic significance, and provides a target for ther-
apeutics (Lengauer et al., 1998); an example is the amplification of HER-2/neu in
breast cancers. At the cytogenetic level, gene amplifications can be detected as
homogeneously stained regions or double minutes. At the molecular level, multiple
copies of an amplicon containing a growth-promoting gene can be detected. The
amplification of N-myc oncogene that occurs in about one-third of advanced neu-
roblastomas is a good example of tumor progression (Seeger et al., 1985).
Almost all solid tumors are genetically unstable. Translocations and gene amplifica-
tions add to the chromosomal abnormalities and may reflect additional mechanisms
for generating genetic instability that occurs as tumors grow. Genetic instability is the
cause of both tumor progression and tumor heterogeneity. As a result, no two tumors are
exactly alike and no single tumor is constituted of genetically identical cells. Tumor
14 Chapter 1
TUMOR HETEROGENEITY
Histological Microdissection
Tissue heterogeneity of histological specimens is well known. Not only neoplastic cells
are heterogenous; a tumor may contain a variable admixture of stromal cells, inflammatory
infiltrates, endothelial cells, and preexisting tissue. This complexity hinders the study of
molecular genetic alterations. Such studies require precise correlation of molecular genetic
characteristics to well-defined cell populations. The presence of multiple cell types close to
one another in the tumor may limit the precise significance of changes in specific cells. As a
result, even sophisticated techniques become less useful when applied to bulk tissue.
Study of uniform cell populations is a prerequisite to understanding differential gene
expression in tumors. A number of mechanical techniques for microdissection have been
developed to isolate cells for analysis from histological sections (Turbett et al., 1996;
Youngson et al., 1995; Going and Lamb, 1996; Moskaluk and Kern, 1997; Lee et al., 1988;
Zhuang et al., 1995). The most sophisticated technique is laser-assisted and suitable for
microdissection of single cells with minimal risk of contamination (Becker et al., 1997).
Some of the microdissection methods are satisfactory but also have certain disadvantages;
for example, laser-assisted techniques require expensive equipment.
To overcome some of the limitations of microdissection methods presently in
use, recently a mechanical technique was developed by Harsch et al. (2001). This device
Introduction 15
ANTITUMOR VACCINES
MOLECULAR GENETICS
detection of DNA, RNA, and protein targets in each specimen on the array. Moreover, con-
secutive sections allow rapid analysis of hundreds of molecular markers in the same set of
specimens. Another advantage is that sufficient cDNA for hybridization to a microarray
can be produced from as little as 1 mg of tissue.
This technology can be used to profile complex diseases and discover novel disease-
related genes. It can dissect complex human diseases by analyzing the pattern of gene
expression. The cDNA microarray method could provide new targets for drug develop-
ment and disease therapies and thus facilitate improved treatment of chronic diseases that
are challenging because of their complexity. Listed below are examples of diseases whose
molecular characteristics have been determined using gene arrays.
Ljubimova et al. (2001) have used 11,000 gene microarrays for identifying gene
expression profiles in brain tumors, including high-grade gliomas (glioblastoma multiforme
[GBM] and anaplastic astrocytoma), low-grade astrocytomas, and benign extraaxial brain
tumors (meningioma), and then compared them with normal brain tissue. In this study the
gene array method was combined with reverse transcriptase (RT)-PCR and immunohisto-
chemical evaluation of glial tumors. All GBMs overexpressed 14 known genes, whereas
these genes were barely detectable in normal human brain tissue. This study also showed that
laminin-80–containing GBMs recurred significantly sooner after surgical removal than did
GBMs with a predominant expression of laminin-9. Thus, overexpression of laminin-8 in
tumor blood vessel walls may be an indicator of time to recurrence for patients with GBM.
Another example of the involvement of many different genes in a cancer is renal cell
carcinoma. This cancer is one of the 10 most frequent malignancies in western countries.
Genes involved in the initiation and progression of this cancer include the von
Hippel–Lindau gene on chromosome 3p, the epidermal growth factor receptor gene on
chromosome 7p, the transforming growth factor gene on chromosome 2p, and the c-myc
oncogene on chromosome 8q (Siezinger et al., 1988; Moch et al., 1998; Lager et al., 1994;
Yao et al., 1998). Other genes involved in renal cancer are currently not known.
Moch et al. (1999) have combined tumor arrays and cDNA arrays for rapid identifi-
cation of genes and their role in renal cell carcinoma. They constructed a kidney cancer
tissue array consisting of 532 renal tumors, 386 of which had clinical follow-up data avail-
able. There were 89 differentially expressed genes in the cancer cell line CRL-1933, one
of them encoding for vimentin. Vimentin expression was significantly associated with poor
patient prognosis independent of grade or stage.
cDNA microarray technology has also been used for verifying the involvement of a
number of genes in another complex disease, rheumatoid arthritis (Heller et al., 1997). In
this disease inflammation of the joint is caused by the gene products of many different cell
types present in the synovium and cartilage tissues plus those infiltrating from the circu-
lating blood. In this study the presence of gene products, such as matrix degrading metal-
loproteinase (MMP), macrophage inflammatory protein (MIP), and human matrix
metalloelastase (HME), was verified. The expression profiles of the genes demonstrate the
utility of the microarrays in determining the hierarachy of signaling events.
The downstream effects of both PAX3 and PAX3-FKHR on NIH 3T3 cells with
cDNA microarrays has also been monitored (Khan et al., 1999). This study elucidated the
pattern of gene expression induced by these two oncogenic transcription factors in these
cells; these factors showed significant myogenic properties. Other recent examples of the
application of gene arrays for determining the molecular parameters of individual tumors
20 Chapter 1
are ovarian and cervical cancers and metastatic versus primary breast cancer (Ono et al.,
2000; Shim et al., 1998; Nacht et al., 1999). New subclasses of leukemia have also been
identified using gene arrays, which have become critical for the successful treatment of
patients (Golub et al., 1999).
Because the individual arrayed tissue samples are very small (0.6 mm in diameter),
one might ask if these specimens are representative of their donor tumors! To answer this
question, Nocito et al. (2001) studied a set of 2,317 bladder tumors that had been previously
analyzed for histological grade and Ki-67 labeling index. The histological grade and the
Ki-67 labeling index were determined for every arrayed tumor sample. The grade and
Ki-67 information obtained on minute arrayed samples were highly similar to the data
obtained on large sections. On the basis of this evidence, it can be stated that intratumor het-
erogeneity does not significantly affect the ability to detect clinicopathological correlations
on the tissue microarrays. It is concluded that tissue microarray is an important tool for
rapid identification of biological or clinically significant molecular alterations in tumors.
ANGIOGENESIS
normal tissue function. It should be noted that tumor angiogenesis is not sufficient to cause
tumor spread and patient death. Tumor cells must also proliferate, penetrate host tissues
and vessels, survive within the vasculature, escape the host immune system, and then begin
growth at a new body site (Weidner, 1998). Before discussing the role of angiogenesis in
disease, it is relevant to explain the process of angiogenesis.
The complex process of angiogenesis includes the recruitment of nearby endothelial
cells, their activation, degradation of the vascular basement membrane, proliferation and
form a new capillary (Albini et al., 2000). Tumor-induced endothelial cell activation leads to
the acquisition of a phenotype characterized by chemotactic motility, basement membrane
invasion, and proliferation. These events are followed by differentiation into a new vessel.
During the last decade there have been significant advances in the understanding of
functional mechanisms of the molecules involved in angiogenesis. Angiogenesis is medi-
ated by multiple positive and negative regulator molecules released by tumor cells, intratu-
moral macrophages, mast cells, and endothelial cells. The balance of the effects of these
mediators determines the outcome of this process. At least three groups of extracellular sig-
nals are involved in angiogenesis: (1) soluble growth molecules such as acid and basic
fibroblast growth factors and vascular endothelial growth factor (discussed later) that affect
endothelial cell growth and differentiation; (2) factors such as transforming growth factor
and angiogenin that inhibit proliferation and enhance differentiation of endothelial cells; (3)
extracellular matrix–bound cytokines released by proteolysis, which contribute to angio-
genic regulation. Other growth factors implicated in different steps of angiogenesis are
platelet-derived growth factor, hepatocyte growth factor, and angiopoietins 1 and 2. Also,
various endothelial surface molecules, such as CD31, CD144, and integrins, play a
role in angiogenesis.
Some of the above-mentioned secreted factors are angiogenic, whereas others are
angiostatic. Thus, angiogenesis is mediated by multiple positive and negative regulatory
molecules released by both tumor cells and the surrounding normal cells. The balance
between these regulators determines whether or not neovascularization will occur. Indeed,
antiangiogenic therapy is based on the use of negative regulators of neovascularization
aimed at suppressing the proangiogenic signal or increasing the inhibitory signals. Albini
et al. (2000) have used the gene therapy approach using class I interferons for effectively
inhibiting tumor angiogenesis and growth of vascular tumors.
Although overwhelming evidence indicates that endothelial cells are central to the
angiogenic process, the following discussion proposes the role of tumors in the formation
of blood vessels. According to Maniotis et al. (1999) and Folberg et al. (2000), blood ves-
sels of malignant eye tumors known as uveal melanomas are formed by tumor cells instead
of endothelial cells. These highly aggressive and metastatic cells are capable of forming in
vivo and in vitro vascular channels, which consist of a basement membrane that stains pos-
itive with the periodic acid–Schiff (PAS) reagent in the absence of endothelial cells and
fibroblasts. The generation of such channels is termed vasculogenic mimicry. This evidence
suggests that angiogenesis may not be the only mechanism responsible for creating tumor
microcirculation. Therefore, methods used to identify the tumor microcirculation by stain-
ing endothelial cells may not be applicable to tumors that express vasculogenic mimicry.
However, certain aspects of the concept of vasculogenic mimicry have been
questioned by McDonald et al. (2000). They indicate that PAS-stained channels do not
22 Chapter 1
represent the microvascular architecture, and endothelial cell–lined blood vessels are also
present in uveal melanomas. They moreover report that tumor cell-lined vessels are infre-
quent in these melanomas. Nevertheless, tumor cells can acquire a new phenotype and par-
ticipate in the formation of blood vessels. It is concluded that the extent and the
pathophysiological significance of cancer cells becoming lining cells and participating in
the formation of blood vessels in tumor is still unclear.
Information on the angiogenesis regulatory molecules has produced new therapeutic
strategies for suppressing angiogenesis and tumor growth or promoting angiogenesis against
coronary and peripheral ischemia and stimulation of wound healing (Thompson
et al., 1999; Kahn et al., 2000). Limited space does not allow discussion of these aspects of
angiogenesis, except for VEGF, which is the most potent angiogenic factor (see pages 23–24).
Angiogenesis plays an important role in the development and progression of a num-
ber of disease states, including various cancers, diabetic retinopathy, macular degenera-
tion, psoriasis, and rheumatic arthritis. The tumor microcirculation plays a key role in
hematogenous dissemination of cancers. There is compelling evidence that angiogenesis
is indeed critical for tumor growth progression and metastasis because tumors require new
blood vessels to achieve a size larger than 2–3 mm. Also, a considerable amount of evi-
dence suggests that tumor angiogenesis is crucial for the growth of solid tumors in vivo
(Folkman, 1996). The growth of tumors (including solid tumors) must be preceded by an
increase in capillaries and newly formed blood vessels that provide tumor cells with oxy-
gen and nutrients as well as paracrine mediators. The blood vessels also remove waste
products.
A large number of tumor blood vessels increases the opportunity of the tumor cells to
enter the circulation. In fact, the newly formed capillaries usually have a fragmented base-
ment membrane, facilitating easier invasion. In the prevascular phase, with little or no
angiogenic activity, the tumor is unable to expand beyond a few cubic millimeters, but
once angiogenic factors are released in sufficient number, the onset of angiogenic activity
stimulates rapid expansion of the tumor.
The microvessel density of the tumor mass, a measure of tumor angiogenesis,
correlates with metastasis and can be used as an independent prognostic factor in the
management of cancer (Jacquemier et al., 1998). A number of studies indicate that
microvessel density gives prognostic information on breast cancer (Weidner et al., 1993).
With respect to prognostic carcinoma, it is thought that a low vascular density correlates
with significantly longer survival duration than with carcinomas having high vascular den-
sity. Thus, neovascularization has proven to be an independent predictor of pathological
state in prostatic carcinoma. A correlation between the endocrine differentiation and
increased neovascularization in prostatic cancer has also been reported (Grobholz et al.,
2000). High-grade tumors with a high neuroendocrine differentiation and increased neo-
vascularization indicate high risk and unfavorable outcome.
Although the role of angiogenesis as a prognostic factor has been most widely
analyzed in breast cancer, angiogenesis also plays an important prognostic role in other
carcinomas such as gastric cancer. This cancer is a highly aggressive malignancy with poor
prognosis and low survival rates. Sanz-Ortega et al. (2000) have evaluated advanced gas-
tric cancers for the expression of oncogenes HER-2/neu (c-erbB-2), c-myc, and epidermal
growth factor receptor, as well as microvessel density. Avidin-biotin immunohistochem-
istry using CD34 stained paraffin sections has shown that tumor angiogenesis is the most
important independent prognostic indicator to predict overall survival.
Introduction 23
The vascular endothelial growth factor (VEGF) is a member of the six-member VEGF
family: VEGF, placenta growth factor, VEGF-B, VEGF-C, VEGF-D, and VEGF-E. These
24 Chapter 1
members have overlapping but specific roles in the growth of new blood vessels. The fol-
lowing discussion is limited to only one member, VEGF (also known as vascular perme-
ability factor), which is the most important and most frequently studied angiogenic factor.
It is a homodimeric 34–42 kDa glycosylated heparin-binding glycoprotein. Alternative
exon splicing of the VEGF gene produces multiple species of mRNA, which encode dif-
ferent VEGF protein isoforms having subunit polypeptides of 121, 145, 165, 189, or 206
amino acid residues (Neufeld et al., 1999).
Vascular endothelial growth factor has three receptors [VEGFR-1 (Flt-1), VEGFR-2
(KDR/Flk-1), and VEGFR-3 (FLT-4)], each consisting of seven immunoglobulin-homology
domains, a transmembrane sequence, and an intracellular portion containing a split kinase
domain (Shibuya, 1995). Ligand (VEGF) binding induces receptor dimerization and sub-
sequent auto/transphosphorylation. The receptors have distinct roles in vasculogenesis and
angiogenesis during embryonic development. Precise roles of the three receptors have
been discussed by Veikkola and Alitalo (1999).
Transcription of VEGF mRNA is induced by a variety of growth factors and cytokines,
including platelet-derived growth factor-BB, epidermal growth factor, tumor necrosis
transforming growth and (Ferrara and Davis-Smyth,
1997). Tissue oxygen tension tightly regulates VEGF levels, and exposure to hypoxia
rapidly and reversibly induces VEGF expression through both increased transcription and
stabilization of the mRNA (Levy et al., 1996). Hypoxic upregulation of VEGF thus provides
a compensatory mechanism by which tissues can increase their oxygenation through induc-
tion of blood vessel growth. Normoxia downregulates VEGF production and leads to
regression of certain newly formed blood vessels. By these opposing processes the vascu-
lature becomes matched to the tissue oxygen demands (Veikkola and Alitalo, 1999).
The vascular endothelial growth factor is produced by tumor cells, macrophages, and
endothelial and smooth muscle cells. It induces vascular endothelial cell migration,
enhances vascular permeability, and promotes extravasation of plasma proteins from tumor
vessels to form an extracellular matrix, facilitating inward migration of endothelial cells
(Callagy et al., 2000). These characteristics impart selectivity to VEGF for endothelial cells.
The vascular endothelial growth factor is involved in angiogenesis in a wide variety of
biological systems, including the female reproductive cycle, wound healing, and tissue
repair. Proliferation of blood vessels during the formation of the corpus luteum in the ovary
and during the growth of endometrial vessels in the uterus occurs upon expression of the
VEGF mRNA and protein (Ferrara and Davis-Smyth, 1997). This factor is also detected
during angiogenesis occurring at the site of embryo implantation in the uterus (Shweiki
et al., 1993). In ischemic cardiac tissue, VEGF mRNA is increased, suggesting the involve-
ment of this factor in the growth of collateral blood vessels (Hashimoto et al., 1994).
In addition to its role in physiological angiogenesis, VEGF is active in pathological
neovascularization. For example, squamous cell carcinoma of the skin strongly expresses
VEGF (Weninger et al., 1996). In fact, tumoral VEGF correlates with prognosis in a vari-
ety of tumors, including breast cancer and malignant mesothelioma (Fig. 1.6).
edge of the tumor) are fixed with formalin and embedded in paraffin. Sections thick)
are mounted onto adhesive-coated slides, dried, and then deparaffinized. The sections are
treated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase activity. After
washing in PBS, the sections are placed in 10 mM sodium citrate buffer (pH 6.0) and boiled
for 5 min in a microwave oven to unmask the antigens. They are washed in Tris buffer
sodium chloride (25 mM Tris-HCl [pH 7.6] and 150 nM sodium chloride) and incubated in
normal goat serum (diluted 1:10 with TBS) for 30 min to block nonspecific staining.
The sections are incubated in the rabbit polyclonal anti-VEGF (Santa Biotechnology,
CA) and diluted 1:100 for 30 min at room temperature. Antigen-antibody reaction is
detected using the biotin-streptavidin–based detection kit (Dako). The reaction is devel-
oped using DAB+hydrogen peroxide and counterstained with Mayer’s hematoxylin.
Exclusion of the primary antibody serves as a negative control. The results of this proce-
dure are shown in Fig. 1.7.
Telepathology (Telemedicine)
Telepathology, introduced by Weinstein et al. (1987), is a pathology practice that
requires telecommunication technologies to transmit digital images to distinct sites for diag-
nostic, consultation, and educational purposes. Telepathology is an affordable option in
places where a pathologist is unaffordable, such as rural hospitals that are too small to sup-
port a pathologist. In addition, telepathology facilitates seeking a second opinion on a diffi-
cult case. It is thought to be accurate and cost-effective, and its advantages outweigh the
problem of waiting longer to have a slide read. Also, the resolution obtainable with videomi-
croscopy is thought to be adequate and appropriate for diagnosis. Telepathology is expected
to become an integral part of medical practice for practical, economic, and humane reasons.
26 Chapter 1
Telepathology can be divided into two major modalities: static imagery and dynamic
(real-time) imagery. Each of these two methods has advantages and limitations. Static
imagery (the store-and-forward method) involves capturing of still images from a micro-
scope and transmitting them through a point-to-point connection or by transmission con-
trol protocol/internet protocol. The still digital images selected at the remote site are
transmitted at a later time for remote diagnosis. The images provided are of superior qual-
ity, but the number of images is limited. It is usually not feasible to transmit the images in
real time, and the selection of images by the remote site requires two pathologists to share
the interaction. The images can be transmitted over the Internet because bandwidth (role
of data transmission) requirements are low for the static imagery.
In static imagery, expedient delivery of high-resolution images can be achieved by
attaching pathology images with an electronic mail message. This method is applicable when
real-time consultation is not required. Despite the low cost and simplicity of static imagery,
Introduction 27
it has certain shortcomings. Because static imagery uses relatively low-resolution digital
photomicrography, it requires high optical magnification to allow adequate examination of
diagnostic images. This results in the need to collect and transmit multiple image files.
Furthermore, static image acquisition means that fields for imaging are preselected by
a person other than the telepathology consultant, leading to unattended field selection
biopsy error (Weinstein et al., 1997). However, high-resolution digital scanning cameras
allow the acquisition of digital images up to 3,400 × 2,700 pixels of resolution. These
images can be captured at a relatively low optical magnification and digitally magnified
multiple times without visible degradation. They can be scrolled at different magnifica-
tions in computer, simulating light microscopy. This high-resolution digital photomicrog-
raphy and the Internet have been used for telepathological gastrointestinal biopsy
consultations (Singson et al., 1999).
In contrast to static imagery, in dynamic imagery the consultant examines a histological
or cytological slide from a remote site by using sophisticated robotic microscopes that trans-
mit real-time digital images through fast and expensive telecommunication links that provide
very high band widths. According to Weinstein (1996), low-resolution dynamic images are
more useful to a pathologist than high-resolution static images. The diagnostic accuracies for
static imaging and dynamic imaging are 88% and 96–97%, respectively. The latter range falls
within the acceptable range for surgical pathology. Although the real-time telepathology
shows a higher diagnostic accuracy, static imagery continues to be the dominant method
used. Static imaging is adequate in those cases where tissue sampling is not a problem.
Attempts have been made to develop systems that combine the advantages of static
imagery and dynamic imagery. Such systems have been described by O’Brien et al.
(1998). However, few of these systems have been implemented because of their complex-
ity. Recently, a new hybrid telepathology system has been described, which achieves
dynamic real-time microscopic video transmission for providing dynamic imaging. The
implementation of this system is awaited.
Imaging standards remain an issue in telepathology. Lack of critical literature in this
field is also a barrier to further development and acceptance of this technology. Furthermore,
standards must be developed and accepted for the types of cases that will be diagnosed and
for protecting patients’ privacy. In addition, telepathology equipment is more expensive than
teleradiology equipment.
The most extensive and well-known telepathology service is part of the U.S. Armed
Forces Institute of Pathology. This service offers the diagnostic evaluation of microscopic still
images sent via e-mail (http://www.afip.org/). Recently, telemicroscopy via Internet browsers
such as Netscape Navigator and Internet Explorer was introduced by Wolf et al. (1998a, b).
They reported a new concept in Internet functionality by demonstrating how Internet
browsers with Java support can use remote control of computer-controlled devices such as an
automatic microscope. More recently, Petersen et al. (2000) reported how this technology can
be used for image transfer and communication between pathologists or research scientists.
Essentially, it is based on a conventional light microscope with a video camera, which in turn
is connected to a computer with a frame grabber and Internet access. This Telemic system
allows the user to show and discuss microscope images with any pathologist who is con-
nected to the Internet. For inquiries about the software and information on the installation, the
reader should contact the Telemic homepage at http://amba.charite.de/telemic
28 Chapter 1
FUTURE OF IMMUNOHISTOPATHOLOGY
cellular physiology and pathology, thus improving our understanding and treatment of
diseases.
I am confident that with the approach of the postgenome era, an ever-increasing num-
ber of human genes will be discovered and their functions elucidated. Combined with the
knowledge of human gene polymorphism, genotyping will allow prediction of the genetic
predisposition to certain diseases, such as cancer. The new millennium will usher us in a
new era of disease-predictive medicine.
PREPARATION OF BUFFERS
ANTIGENS
An antigen is a substance that reacts specifically with receptors on the surface of lym-
phocytes and with their soluble products such as antibodies. Antigens usually are large,
complex protein or polysaccharide molecules with molecular weights usually greater than
31
32 Chapter 2
40,000. However, the molecular weight, for example, may vary from 15,000 (hen egg white
lysozyme) to 2,000,000 (keyhole limpet hemocyanin) daltons. Protein antigens function as
the most potent immunogens, and polysaccharide antigens rank second. For cell-mediated
immunity, only proteins serve as immunogens. Certain nucleic acid types such as Z-DNA
and other molecules can also stimulate antibody production.
Antigens can be defined on the basis of four immunological properties: immuno-
genecity, antigenicity, allerogenicity, and tolerogenicity. The ability of a substance to
induce an immune response is called immunogenicity. Most antigens have a variety of
different antigenic determinants (epitopes) on their surfaces, which stimulate antibody
production. Antigenicity is the ability of an immunogen to combine with an antibody or
cell surface receptors.
Allerogenicity is the ability to induce various types of allergic responses. Allergens
are immunogens that tend to activate specific types of humoral or cell-mediated responses
having allergic manifestations (Kuby, 1992). Tolerogenicity is the capacity to induce spe-
cific immunological nonresponsiveness in either the humoral or the cell-mediated systems.
In other words, experimentally induced tolerance can be defined as a state in which an ani-
mal fails to respond to an antigen that would normally be immunogenic. It is not known
whether similar mechanisms generate both naturally acquired self-tolerance and experi-
mentally induced tolerance.
Immunogens induce an immune response only if they are recognized as foreign (non-
self). A case in point is protein bovine serum albumin, which is immunogenic in sheep but
not in cows. Most large antigens have multiple reactive sites, or epitopes, on their surfaces,
which can induce production of specific antibodies. Antibodies do not recognize the whole
immunogen but only small regions (epitopes). Each type of antibody binds to its own
inducing epitope. For example, lysozyme, an enzyme that degrades the carbohydrate coat
of bacteria, induces several different antibodies, each of which binds to a particular epi-
tope on the lysozyme molecule (Lodish et al., 2000). Although different epitopes on
lysozyme differ greatly in their chemical properties, the interaction between lysozyme and
antibody is complementary in all cases. In other words, the surface of the antibody’s antigen-
binding site fits into that of the corresponding epitope as if they are molded together. The
intimate contact between these two surfaces, stabilized by numerous noncovalent bonds,
is responsible for the exquisite binding specificity shown by an antibody.
Epitopes
remaining part of the protein antigen molecule is the carrier for the epitope. An antigen such
as bovine serum albumin has several different epitopes on its surface, each of which stimu-
lates the cell having the appropriate receptor. Because an epitope usually comprises a
sequence of approximately three to eight amino acid residues, antibodies should be regarded
as site- or region-specific detection molecules instead of antigen-specific molecules.
ANTIBODIES
Immunohistochemical labeling with antibodies has become the most sensitive and pow-
erful method for localizing antigens in situ and thus for characterizing cells and their com-
ponents and their functions. In this context the importance of antibody specificity and
selectivity for the antigen cannot be overemphasized. The specificity relies entirely on the
properties of the primary antibody, independent of the procedure used for detection. Although
both monoclonal and polyclonal primary antibodies can be generated or purchased, the for-
mer are preferred and are in much wider use because of their far greater specificity.
The basic structure of an antibody molecule is Y-shaped, with the two tips designed
to recognize and bind antigens (Fig. 2.1). The tips, through the disulfide bridge, are free to
bend with respect to each other. This property increases the binding strength of the anti-
body for antigens that have multiple, adjacent antigenic determinants and for antigens that
are closely packed together. The remainder of the antibody molecule enables it to interact
with other proteins, preventing undesirable company.
Antibodies are molecules secreted by terminally differentiated B cells (a type of lym-
phocyte) known as plasma cells. Nearly all rabbit primary antibodies and most mouse
monoclonal antibodies are immunoglobulins (Igs). There are five classes of Igs that differ
structurally and functionally. Immunoglobulin G (IgG) molecules are the major class of
Igs in the blood, which are predominantly produced in the secondary immune response.
Monoclonal antibodies have been termed magic bullets and hailed in publications as the
cure for cancer. Belief in this idea was strengthened by the successful clinical results of mouse
34 Chapter 2
Polyclonal Antibodies
Both polyclonal and monoclonal antibodies have advantages and limitations with
regard to their generation, specificity, cost, and overall applications. Polyclonal antibodies
possess higher affinity and wider reactivity but lower specificity. They have the advantage
of detecting many types of epitopes and recognizing antigens of different orientations.
Polyclonal antibodies show greater stability at varying pH levels and salt concentrations
and are more useful for preadsorption controls. They are simpler to produce in a shorter
duration, and there is no risk of loss of clones. In addition, large animals (e.g., rabbits and
horses) can be used to recover large volumes of antibody-rich serum. However, a fresh
batch of the serum is required when the original stock is exhausted. This replacement
results in batch-to-batch variation, which may result in differences in antibody reactivity
and titer (Nelson et al., 2000). Such differences result in a lack of reproducibility.
Polyclonal antibodies are composed of multiple species of immunoglobulins directed
toward several epitopes within a particular antigenic molecule. Moreover, only a minor
proportion of the antibody present in the polyclonal antiserum is specific for the immu-
nizing antigen. The remainder may consist either of antibodies produced by the animal in
the past in response to previous antigenic stimuli or of antibodies against contaminating
antigens present in the immunizing preparation (Mason et al., 1983). Even the antibodies
in the polyclonal antiserum that are specific for the immunizing antigen are usually het-
erogeneous and are directed against a number of different epitopes on the immunizing anti-
gen.
However, although whole sera or whole IgG fractions of polyclonal antibodies often
have problems, affinity purification against an antigen affinity column can dramatically
improve the usefulness of the polyclonal reagents. Thus, a mixture of isoforms of antibod-
ies to different epitopes is obtained. These epitopes are still relatively unique to the antigen
involved. The main advantage is that 100% of the antibody in these preparations reacts with
the antigen, often at multiple and therefore additive sites. This approach is analogous to
mixing monoclonal antibodies to label different epitopes together. Although such reagents
Antigens and Antibodies 35
Affinity Chromatography
Antibody affinity chromatography is employed to isolate antigen-specific antibodies.
The most common affinity matrix for coupling of molecules is cyanogen bromide–activated
36 Chapter 2
Sepharose. The following procedure can be used to purify antibodies raised against a
particular protein (Javois, 1999).
can be concentrated and stored at 4°C for weeks or at – 80°C for months and
years. Avoid repeated freezing and thawing, which may denature the proteins.
Monoclonal Antibodies
Unlike polyclonal antibodies, monoclonal antibodies are directed against single epitopes
consisting of very short sequences of amino acids. These antibodies are able to provide invalu-
able information about the molecular conformation of a particular epitope within a given anti-
gen. However, even though a monoclonal antibody can recognize multiple molecules, its
specificity remains intact. This phenomenon is due to the presence of very similar epitopes in
different peptides and proteins. This subject is discussed in detail later in this chapter.
The development of monoclonal antibodies has led to a new era of enhanced diagnos-
tic and therapeutic modalities. The impact of this development on diagnostic imaging tech-
nologies has been dramatic, resulting in a revolution in modern diagnostic medicine. Recent
advances in recombinant antigen preparation have further advanced the usefulness of these
methods; almost any protein can be engineered to be expressed in nonnatural host cells.
If monoclonal antibodies to a specific region or a specific epitope of an antigen are
desired and the amino acid sequence of this region is known, synthetic peptides can be pre-
pared for animal injection. In fact, synthetic peptides are currently being used for the injec-
tion of animals to generate antibodies for a specific region or a specific region epitope of
an antigen. As a result, monoclonal antibodies of high affinity and specificity are being
produced at a fast pace. These antibodies are being effectively employed for the detection
and analysis of antigens, and are playing a key role in clinical diagnostic medicine. These
developments are partially responsible for the exponential growth in our understanding of
the physiology of human diseases.
Monoclonal antibodies are produced only when necessary because their generation is
difficult, time-consuming, and frustrating. Nevertheless, the most important reason for pre-
ferring them is their exquisite specificity, as ideally they recognize only one type of epi-
tope. Moreover, these antibodies show a high biological half-life in blood and other
tissues, rendering them effective for prophylactic use. The toxicity of infused monoclonal
antibodies is expected to be low because of their biological nature. Thus, these antibodies
are being used extensively and successfully in routine pathology laboratories to aid in the
clinical diagnosis and treatment of malignant diseases.
To produce monoclonal antibodies, lower doses of antigens are required for
immunoresponse. The continuous culture of B cell hybridomas yields a reproducible and
potentially inexhaustible supply of the monoclonal antibody; all batches are homogenous.
Consequently, these antibodies allow the development of standardized procedures for
clinical diagnosis. In addition, monoclonal antibodies are ideal for a complex mixture of
antigens (e.g., membrane antigens), for scarce antigens, or when attempting to detect
unique epitopes where purification is difficult or impossible (Beltz and Burd, 1989).
Monoclonal antibodies do not have high affinities and so generally must be used at lower
dilutions. Moreover, monoclonal antibodies are more expensive to generate or purchase
than polyclonal antibodies. Exceptions to some of the advantages and limitations of
polyclonal and monoclonal antibodies listed above are not uncommon. A large number of
monoclonal antibodies are commercially available (see page 50).
38 Chapter 2
noted, however, that the major effect of aldehyde fixation on the reaction of the antibody
with the antigen is the inaccessibility of the antigen to the antibody. In other words, the
crosslinking of cytosol proteins introduced by aldehyde fixatives creates a barrier for the
antibody molecule to penetrate the cell and react with the antigen (epitope).
Monoclonal antibodies generated against epitopes react with the native conformation
of the antigen, which may be preserved by fixation. These antibodies are useful for
immunohistochemistry, provided antigens are accessible to antibodies. In contrast, some
other monoclonal antibodies preferentially react with denatured antigens after treatment
with denaturing agents such as sodium dodecyl sulfate (SDS). Such antibodies generated
by immunizations with isolated peptides usually are not well suited for immunohisto-
chemistry (Willingham, 1999). Based on these observations, it is likely that most mono-
clonal antibodies are reactive under certain appropriate conditions used to prepare and
incubate the specimens of interest. There are still other antibodies that are reactive with
both native and denatured conformations of proteins. It is thought that these antibodies rec-
ognize peptide sequences exposed when proteins are denatured. On the other hand, poly-
clonal antibodies do not require optimal processing condition because a polyclonal
antibody recognizes multiple epitopes. These antibodies may recognize an epitope other
than that under study.
but not in all, a correlation also exists between the label index and patient survival.
However, although these indices are related, clinical comparison is necessary to determine
which is the better prognostic marker for human breast cancer and other human
cancers.
A brief comment on the usefulness of the BrdU method is relevant. Currently, immuno-
histochemical detection of exogenously injected 5-bromodeoxyuridine several hours prior to
animal sacrifice is widely used to assess proliferation state in murine tissues. This nucleotide-
analog probe is integrated into the DNA of replicating cells during S phase (Selden et al.,
1993). The probe can be subsequently detected immunohistochemically in paraffin-embedded
tissue sections. However, the proportion of anti-BrdU stained cells is variable depending on
the duration and frequency of BrdU injections before sacrificing the animal.
A number of examples given below support the prognostic value of MIB-1 antibody.
Evaluation of proliferation index in malignant mesothelioma can be performed using the
MIB-1 antibody (Comin et al., 2000). In this case a correlation is found between the label-
ing index and survival. Malignant mesothelioma is a rare, aggressive, and frequently lethal
tumor, usually associated with asbestos exposure. It should be noted that numerous prog-
nostic factors (e.g., age, tumor stage, asbestos exposure, performance status, histological
subtype, tumor angiogenesis, and proliferation index) are correlated with survival. Another
example is the high proliferative index found in the esophageal small cell carcinoma using
MIB-1 antibody, indicating aggressive behavior (Lam et al., 2000).
Also, Ki-67 antigen labeling with MIB-1 antibody is a reliable method for estimating
the proliferative activity in uveal melanomas after proton beam irradiation (Chiquet et al.,
2000). The Ki-67 score is significantly correlated with prognostic variables (mitotic index
and histological largest tumor diameter) and with radiation effects after proton beam irra-
diation. Furthermore, a higher Ki-67 score is found in uveal melanomas with metastasis
than in tumors without metastatic evolution. Uveal melanoma is the most common primary
adult ocular malignancy. Their ability to metastasize is well recognized. It should be noted
that these views have not been accepted universally.
Another example is the labeling of Ki-67 with MIB-1 antibody in benign and malig-
nant apocrine lesions of the breast, which facilitates differentiation between benign and
malignant breast apocrine lesions (Moriya et al., 2000). Both the number of positive cases
and the percentage of positive tumor cells are significantly higher in malignant cases than
in benign apocrine cases. Thus, Ki-67 immunohistochemistry can be an auxiliary method
for determining the possible biological behavior of lesions and for the diagnosis and prog-
nosis of patients with apocrine lesions of the breast.
MIB-1 immunostaining in conjunction with microwave antigen retrieval is a benefi-
cial adjunctive test when the morphological features are suggestive but not diagnostic for
vulvar condyloma acuminatum (Pirog et al., 2000). Histopathological confirmation of
condyloma acuminatum implies human papillomavirus (HPV) infection, which is sexually
transmitted and confers an increased risk for synchronous or subsequent HPV-associated
wartlike lesions elsewhere in the female genital tract. However, histopathological diagno-
sis of condyloma acuminatum is often based on architectural features that are not specific
for HPV infection.
To avoid this problem, the application of MIB-1 antibody is useful in the differential
diagnosis of benign exophytic vulvar lesions because HPV-associated lesions show
Antigens and Antibodies 41
increased cellular proliferation. Limiting the diagnosis of condyloma to only those lesions
with koilocytotic atypia will result in underdiagnosis, and categorizing equivocal wartlike
lesions of the vulva as c/w condyloma acuminatum is associated with substantial over-
diagnosis. Complete concordance was found between MIB-1 positivity and detection of
HPV by both in situ hybridization and polymerase chain reaction (Pirog et al., 2000);
however, MIB-1 positivity is more sensitive than the other two techniques.
Antibody MIB-1 also shows cross-reactivity. It has been demonstrated, for example,
that this antibody shows strong staining of cell membrane and cytoplasm in hyalinizing
trabecular adenoma of the thyroid gland (Hirokawa and Carney, 2000). In contrast, this
antibody does not show similar immunoreactivity with papillary carcinoma. This informa-
tion provides a morphological difference between hyalinizing trabecular adenoma and
papillary carcinoma, which is important because it has been suggested that these two types
of tissues are closely related tumors (Fonseca et al., 1997). This benign tumor does share
histological features, such as intranuclear cytoplasmic invaginations, nuclear grooves, and
psammoma body–like formations, with papillary carcinoma. Thus, MIB-1 antibody can be
diagnostically useful in differentiating the benign tumor from papillary carcinoma.
Note that MIB-1 antibody fails to react with Ki-67 antigen in the tissue fixed with
formaldehyde or Kryofix in the absence of heat pretreatment. Because Kryofix is a non-
protein, crosslinking fixative, breakdown of protein crosslinkages is not responsible for the
availability of Ki-67 antigen in this case. On the other hand, MIB-1 antibody readily reacts
with Ki-67 antigen in tissue fixed with either of these two fixatives with heat pretreatment.
It is apparent that in this case the effect of heat treatment is due to factors other than break-
down of protein crosslinkages.
Although MIB-1 antibody is a reliable tool for determining proliferating cells in
human tissues, it does not react with the homologous mouse antigen. Therefore, this anti-
body is useless in experimental pathology using mice as the model system. Because the
use of murine tumor models has steadily increased, there is a growing need for a prolifer-
ation marker for routinely processed paraffin-embedded murine tissues. Such a marker is
monoclonal antibody MIB-5, which is raised against bacterially expressed parts of the
human Ki-67 cDNA (Schlüter et al., 1993; Gerlach et al., 1997). Recently, Birner et al.
(2001) have shown that MIB-5 detects Ki-67 antigen in formalin-fixed, paraffin-embedded
murine tissues, equivalent to MIB-1 staining of human tissues.
Coating buffer
3.18g
5.84 g
Distilled water 1,000 ml
Adjust pH to 9.5
Well 11 is skipped and well 12 is coated with of the antigen. Wells in the second
row are filled with of coating buffer to serve as blanks. Incubation is carried out for
30 min at 37°C.
Antigens and Antibodies 43
Diluting buffer
Tween 20 0.5 ml
NaCl 8.5 g
Distilled water 1,000 ml
Adjust pH to 7.2
To raise pH, add 30 ml of to lower pH, add 100 ml of this solution. All
wells in a row are carefully rinsed with a wash buffer simultaneously using a well-washing
device (Nunc Immunowash).
Wash buffer is the same as diluting buffer but without Tween 20.
The rinsing is done at least eight times, and all buffer after the last rinse is removed.
Starting with the last serum dilution, is transferred to well 10 of each row. Then,
of the next serum dilution is transferred to well 9 of each row, and so on until one
has reached the starting dilution, of which is transferred to each well 1. Incubation
is carried out for 30 min at 37°C. This is followed by thorough rinsing with the wash buffer
using a well-washing device as indicated above.
One hundred microliters of commercial goat antirabbit globulin conjugated to horse-
radish peroxidase (diluted 1/500 diluting buffer) is added to every well. Incubation is done
for 30 min at 37°C, followed by washing, and of peroxidase substrate in freshly
prepared substrate buffer is added to each well.
Substrate buffer
11.85g
Citric acid 11.73 g
Distilled water 1,000 ml
Adjust pH to 4.0
Substrate
Ten milligrams of 2,2´-azino-di-(3-ethylbenzthiazoline sulfonic acid) diammonium
salt is dissolved in 50 ml of substrate buffer containing of 30% hydrogen peroxide.
Only freshly prepared substrate should be used. Incubation is carried out for 30 min at 37°C,
followed by visual plate reading on a 1+ to 4+ basis or at if a microtiter plate
reader is available. The optical density of the instrument is set to zero with the antigen con-
trol (well 12 in the first row). Any optical density in the antibody controls (bottom row) is
subtracted from the corresponding test well above.
The proteins are delivered subcutaneously. Regular boosting is needed to augment
polyclonal response, which is monitored using tail bleeds that provide sufficient serum to
make sure the antibody titer to a desired antigen, using the enzyme-linked immunosorbent
assay (ELISA). Boosting also encourages immunoglobulin class switching and the gener-
ation of higher affinity antibodies through somatic hypermutation. Generally, IgG mono-
clonal antibodies are preferred because they are less prone to degradation and more useful
44 Chapter 2
as therapeutic reagents. Antigenically responding B cells are removed aseptically from the
spleen or lymph node to obtain cells for hybridization.
2. Fusion and selection. The murine splenic B cells are fused with histocompatible
myeloma cells such as Sp2/0. The myeloma cells are preselected for a deficiency in the
enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) by culturing in
medium containing 8-azaguanine. The B cells are mixed with HGPRT-negative myeloma
cells and a fusing agent such as polyethylene glycol. The mixing and centrifugation steps
generate myeloma-splenic B cell hybridomas. These hybrid cells are plated into tissue cul-
ture wells. Unfused myeloma cells are removed using a selective medium containing
hypoxanthine, aminopterin, and thymidine (HAT); all unfused myeloma cells will die.
Hybridomas are carefully examined with an inverted microscope. Once established, the
hybridoma colony will continue to grow in the culture medium (such as RPMI-1640 con-
taining antibiotics and fetal bovine serum) and produce antibodies. Approximately 1 month
post fusion, hybridomas can be propagated in HT medium (hypoxanthine and thymidine).
3. Screening. Primary screening is necessary to eliminate nonspecific hybridomas as
soon as possible. Screening is also used to test the hybridoma culture supernatant for anti-
body reactivity and specificity. As an example, an Epstein-Barr virus associated protein is
coated onto plastic ELISA plates. After incubation of hybridoma culture supernatant, sec-
ondary enzyme-labeled conjugate and chromogenic substrate, a colored product indicates
a positive hybridoma. Alternatively, immunocytochemical screening can be used. It is
preferable to test hybridomas when at least three-quarters of them are confluent.
4. Characterization. The reactivity, specificity, and cross-reactivity of the potential
monoclonal antibody can be analyzed by using culture supernatant or a purified immu-
noglobulin preparation. It may be necessary to redone hybridomas by limiting dilution
because the original colony might contain at least two populations of fused B cells. In the
absence of such an analysis, the presence of antibodies of different class, specificity, and
affinity might yield ambiguous results. Characterization also provides the opportunity to
test against a wide panel of related antigens or tissue preparations. This is important espe-
cially for histopathological studies. Once the purification of the hybridoma is established,
bulk production of a monoclonal antibody can be obtained using surface-expanded tissue
culture flasks. It should be noted, however, that although a hybridoma may be the fused
product of a single B cell and produce a monoclonal antibody of refined specificity, such
an antibody in some cases can cross-react with other antigens or exhibit dual or multiple
specificity. The phenomenon of cross-reactivity is discussed on page 48.
It is relevant to explain the use of the letters CD as a prefix to monoclonal antibodies.
Lymphocytes possess many different surface proteins, each of which possesses many dis-
tinct epitopes. To classify these lymphocyte antigens, a numbering system has been estab-
lished that clusters molecules having similar epitopes. Thus, all monoclonal antibodies that
detect the epitopes on a single antigen are assigned to a numbered cluster of differentia-
tion (CD). In most cases a defined CD denotes a protein of specific function. For example,
the protein called CD4 is associated with cells (lymphocytes) that help the immune
response, while CD8 is found on cells that suppress the immune response.
to multimeric antigen with strong avidity, and bispecificity facilitates the crosslinking of
two antigens, for example, in recruiting cytotoxic T cells to mediate killing of a tumor cell.
Bivalent (IgG) antibodies can be derived from hybridomas, and bispecific antibodies by
fusion of two hybridomas with two different specificities. Bispecific antibodies can form
useful immunotherapeutic tools in cancer treatment. The limited space available in this
volume does not allow detailed discussion of the applications of these antibodies.
Important in vivo animal studies, which administer bispecific antibodies, and clinical tri-
als using these antibodies are listed by Koelemij et al. (1999).
Bispecific antibodies have been known for a long time. The first antibody with dual
specificity was described approximately 40 years ago (Nisonoff and Rivers, 1961). The
potential usefulness of these antibodies in the treatment of malignancies becomes clear
when one considers that some therapeutic applications of monoclonal antibodies in
patients with cancer have shown disappointing results. However, in general, monoclonal
antibodies are thought to achieve antitumor effects by inducing antibody-dependent cellu-
lar toxicity or complement-mediated cytotoxicity. Considerable experience has been
gained in using monoclonal antibodies in patients with cancer, especially in the treatment
of hematological malignancies (Matthews et al., 1995). Nevertheless, the application of
many monoclonal antibodies does not completely eliminate tumor cells. The cell surface
expression of complement-deactivating molecules is thought to be the escape mechanism
used by tumor cells in this incomplete elimination.
Because many potentially useful monoclonal antibodies do not possess the appropri-
ate isotype and so are unable to activate human complement and/or trigger on human
cells, treatment strategies are needed. One such strategy is the application of bispecific
monoclonal antibodies that exploit the specificity of monoclonal antibody and ensure
activation of cellular cytotoxic mechanisms (Fanger et al., 1993).
Bispecific monoclonal antibodies are artificially developed antibodies with antigen-
binding sites physically linked to different specificities. It is thought that bispecific mono-
clonal antibodies activate the cellular immune response by crosslinking immune cells to
tumor cells, thus circumventing the proper structures for tumor cell–immune cell interac-
tions (Koelemij et al., 1999). These antibodies are effective in low concentrations in vivo.
For example, Kufer et al. (1996) have combined the anti-CD3 specificity directed against
T cells in a bispecific monoclonal antibody, with the specificity against the tumor-associated
17-1A antigen. This antibody could be a major improvement, for example, in the therapy
for disseminated micrometastatic tumor cells.
Bispecific monoclonal antibodies are being evaluated in phase I and II studies in a
variety of malignant diseases in the fields of hematooncology and solid tumors. It is likely
that in the next decade immunotherapy using bispecific monoclonal antibodies will have a
place, complementary to the current modalities such as surgery, chemotherapy, hormone
therapy, and radiation, in the treatment of malignancies.
the products are heterogeneous, ill defined, and large. Hybrid antibodies were also pro-
duced by chemical processing of Fab fragments to reconstitute (hybrid) dimers (Brennan
et al., 1985). These antibodies are free of mono specific contaminants and thus avoid many
unwanted side reactions.
The most commonly used technique to produce bispecific antibodies from two
monoclonal antibodies is by fusing two hybridoma cell lines by conventional cell fusion
procedure (Staerz and Bevan, 1986). These cells produce all possible combinations of the
heavy and light chains of both antibodies, including the desired bispecific antibody. A
limitation is that only part of the antibodies is the desired bispecific monoclonal antibody;
therefore, further purification is necessary (Van Ravenswaay et al., 1993).
Currently, mostly molecular biological techniques are used to obtain bispecific mono-
clonal antibodies. By focusing on a genetic approach, molecules having the appropriate
binding regions as well as bispecificity can be prepared. For example, a so-called leucine
zipper technique has been employed for producing bispecific monoclonal antibodies
(Kostelny et al, 1992). Leucine zipper peptides of the transcription factors Fos and Jun
preferentially form heterodimers. In a genetic construct the sequences of the tail of one
monoclonal antibody are replaced by the sequences from the leucine zipper region of Fos,
and the sequences of the other monoclonal antibody are replaced by the sequences of the
Jun leucine zipper. The murine myoloma cell line Sp2/0 is transfected with these con-
structs, resulting in the production of heterodimers of Other methods have
focused on obtaining single-chain bispecific molecules. These molecules consist of two
variable domains connected by a polypeptide spacer of two monoclonal antibodies,
coupled by a linker (Traunecker et al., 1992).
Another approach to construct small bivalent antibody fragments is through the
dimeric antibody fragments (diabodies) (Holliger et al., 1993). The diabodies can be eas-
ily obtained from bacteria, can be expressed in high yield, and lack the Fc portions of the
whole immunoglobulin. These antibody fragments with two antigen-binding sites com-
prise a heavy-chain variable domain connected to a light-chain variable domain on the
same polypeptide chain. Using a linker that is too short to allow pairing between the two
domains on the same chain forces the domains to pair with the complementary domains of
another chain and create two antigen binding sites. It should be noted that antibody frag-
ments are often preferable to complete antibodies, as the Fc region of antibodies can lead
to undesirable targeting to cells expressing Fc receptors.
RECOMBINANT ANTIBODIES
A more recent strategy for cancer diagnosis and therapy is the application of geneti-
cally engineered antibodies. This approach is logical because antibodies are the paradigm
for the design of high-affinity protein-based binding reagents. This technology includes
chimeric and humanized antibodies, antibody libraries, and transgenic organisms as biore-
actors (Gavilondo and Larrick, 2000). During the last 10–15 years, important advances
have been made in the design, selection, and production of new types of recombinant anti-
bodies. Recombinant antibodies have been reduced in size, dissected into minimal binding
fragments, and rebuilt into multivalent high-avidity reagents (Hudson, 1999).
Recombinant antibody fragments have also been fused to radioisotopes and with a variety
Antigens and Antibodies 47
of molecules, including enzymes for prodrug therapy, toxins for cancer treatment, viruses
for gene therapy, cationic tails for DNA delivery, liposomes for improved drug delivery,
and biosensor surfaces for cancer diagnosis and therapy for real-time detection of target
molecules.
For clinical diagnostic applications, antibody fragments alone (without Fc) can pro-
vide the full range of in vitro immunoassays through to in vivo tumor-targeting reagents.
In fact, recombinant antibodies and their fragments have entered the clinic, both for can-
cer diagnosis and for therapy. These reagents represent more than 30% of all biological
proteins undergoing clinical trials for diagnosis and therapy. Clinical results confirm that
these new antibodies, directed at the appropriate tumor markers (e.g., CD20 and HER-2),
can control diseases without apparent side effects (Cragg et al., 1999). Innovative selection
methods have enabled the isolation of high-affinity cancer-targeting and antiviral antibod-
ies, the latter capable of redirecting viruses for gene therapy applications (Hudson, 1999).
It is now possible to select high-affinity antibody fragments directly from a viral culture
rather than from a live mouse. One significant advantage of this new technology is the iso-
lation of antibodies with new binding specificities against hitherto refractory antigens, thus
avoiding the limitations inherent in the mammalian immune response (De Haard et al.,
1999). In addition, bispecific antibodies and related fusion proteins have been produced for
cancer immunotherapy, effectively enhancing the human immune response in anticancer
vaccines and T cell recruitment strategies.
More recently, a novel technique has been developed for high-throughput screening
of recombinant antibodies, based on the creation of antibody arrays (De Wildt et al., 2000).
This method uses robotic picking and high-density gridding of bacteria containing anti-
body genes followed by filter-based ELISA screening to identify clones that express bind-
ing antibody fragments. This approach can screen thousands of different antibody clones
at a time against a large number of different antigens. Thus, antibodies against impure pro-
teins and complex antigens can be isolated. However, because a cellular extract contains
thousands of different proteins, the detection sensitivity of this filter-screening technique
requires considerable improvement to be useful for fingerprinting differentially expressed
proteins.
The Food and Drug Administration has approved the use of engineered therapeutic
antibodies. This technology is expected to be an important instrument in the toolbox of the
molecular biologist. Although it is not exactly clear how monoclonal antibodies damage
tumors in vivo, it is thought that the ability of the antibodies to crosslink membrane recep-
tors and generate intracellular signals is part of the mechanism controlling the tumor
growth.
Interest in the use of monoclonal antibodies in diagnosing and treating cancer has
undergone a resurgence during the last decade. The most important reasons for the
renewed interest are (Murray, 2000): (1) the development of human or humanized anti-
bodies through recombinant techniques, resulting in the decrease or elimination of
immunogenicity, (2) approval of monoclonal antibodies (trastuzumab and rituximab) for
48 Chapter 2
cancer treatment by the Food and Drug Administration, and (3) antitumor effects of mon-
oclonal antibodies, especially on solid tumors as shown by clinical trials when used alone
or in combination with chemotherapy or radiotherapy.
Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized
monoclonal antibody that recognizes the human oncoprotein HER-2/neu, which is over-
expressed in some breast cancers and other tumors. Rituximab (Rituxan; Genentech,
South San Francisco, CA, and I DEC Pharmaceuticals, San Diego, CA) is a genetically
engineered chimeric monoclonal antibody containing murine light- and heavy-chain and
kappa light-chain constant regions. It binds to CD20, a differentiation antigen found exclu-
sively on B cells and on more than 95% of B-cell non-Hodgkin’s lymphoma, but not on
hematopoietic stem cells, preB cells, normal plasma cells, or other tissues.
Monoclonal antibodies can mediate tumor destruction by both direct and indirect
mechanisms. Direct mechanisms include (1) increased induction of apoptosis resulting
from binding to calcium channels and (2) inhibition of ligand binding and suppression
of transcription factors within the tumor cells resulting from binding to growth factor
receptors (e.g., EGFR and HER-2). Monoclonal antibodies can also destroy tumor cells
indirectly through immunological mechanisms such as antibody-dependent cell-mediated
cytotoxicity and complement-dependent cytotoxicity (Murray, 2000). The type of antigen
to which the antibody binds is also relevant. The most effective monoclonal antibodies are
those that bind to specific receptors and possess the strongest biochemical and/or immuno-
logical effects.
ANTIBODY CROSS-REACTIVITY
The ability of antibodies to bind molecules other than those molecules used as the
immunogens is well known. Such a binding is termed cross-reactivity. Cross-reactivity is
sometimes a source of confusion in the interpretation of immunohistochemically stained
preparations because it cannot be detected by the usual controls for specificity.
Although the antigen-antibody reaction is highly specific, in some cases even a mon-
oclonal antibody elicited by one type of antigen can cross-react with another type of anti-
gen. In other words, two closely similar proteins may react with an antibody raised by only
one of them. There are a number of reasons for cross-reactivity. This problem arises when
an antibody-combining site recognizes more than one antigenic determinant due to
similarity in shape of different antigens. The cross-reaction can occur when the antigenic
determinant site is a sequence of amino acids common to more than one antigen. This
commonality can occur when the antigenic determinant is conserved in a family of pro-
teins. Many proteins possess homologies in their amino acid sequences and thus show
immunological cross-reactivity.
Cross-reaction can also occur when two different antigens share an identical epitope
or if antibodies specific for one type of epitope also bind to an unrelated epitope possess-
ing similar chemical composition. However, the antibody-binding affinity for the cross-
reacting epitope is usually less than that for the original epitope. Processing conditions can
expose an epitope related to the epitope under study but less so the former epitope. Careful
evaluation of a given monoclonal antibody and its determinant can be accomplished by
epitope mapping (Nelson et al., 1997).
Antigens and Antibodies 49
Another reason for cross-reactivity is the use of less than pure protein or conjugated
or fusion proteins as immunogens; such immunogens produce a heterogenous population
of the antibody having considerable cross-reactivity to the contaminants (Javois, 1999).
Cross-reactivities to the carrier protein to which the antigen has been conjugated or fused
can be removed by affinity chromatography; however, a possible disadvantage of this
method is that the most desirable immunoglobulins having the highest affinity bind the
tightest and are difficult to recover. These problems can be prevented and increased
antibody specificity can be achieved by using synthetic peptides or protein fragments as
eliciting antigens.
Another potential source of cross-reactivity is the presence of Fc receptors in cells or
tissues, which bind the Fc region of the primary or secondary antibodies. These nonspe-
cific sites can be blocked with normal serum or nonimmune immunoglobulins. When a
secondary antibody is used for detection, the normal serum or immunoglobulin for block-
ing should be from the same species as the secondary antibody.
Undesirable or nonspecific staining can also be the result of impure reagents used in
the staining. This background staining can be prevented by using purified reagents and
optimizing conditions for tissue processing including staining. Nonspecific binding can
also occur owing to ionic interactions with other proteins or organelles in the tissue
(Grube, 1980). These interactions can be minimized by diluting the antibody and by
increasing the salt concentrations in the diluent and the rinsing solutions (Javois, 1999).
POLYREACTIVE ANTIBODIES
Polyreactive antibodies are naturally occurring antibodies. They are primarily from
IgM but also from IgG and IgA isotypes. Polyreactive antibodies are capable of reacting
with a variety of antigens that may differ among themselves. Unlike classic autoantibod-
ies, which react with specific host antigens, polyreactive antibodies react with specific host
antigens. Polyreactive antibodies react with endogenous as well as exogenous antigens.
The production of polyreactive antibodies is independent of antigen immunization.
The affinity of these antibodies for different antigens varies but is relatively low compared
with the affinity of monoclonal antibodies elicited by a mature antigen-driven response
(Chen et al., 1995).
It is thought that polyreactive antibodies have physiological relevance. These antibod-
ies probably are involved in defense distinct from their counterpart monoreactive antibodies.
Polyreactive antibodies constitute a first line of defense against invading microorganisms by
enhancing phagocytosis or complement-mediated lysis or by amplifying an ongoing specific
antibody response. In other words, polyreactive antibody–producing/antigen binding cells
might play a role in the development and maintenance of immunological tolerance. It is also
possible that polyreactive antibodies play a role in the homeostasis of all internal biological
systems. These antibodies are known to bind to antigens in the blood and rapidly clear from
the circulation (Sigounas et al., 1994).
Various assays, including ELISA, immunoblots, and chamber ELIspot, have been
employed for demonstrating polyreactivity of preimmune antibodies in both mice and
humans (Ternynck and Avrameas, 1986; Quan et al., 1997; Klimman, 1994). In a complex
biological matrix, polyreactive antibodies are bound by components of the matrix, leaving
50 Chapter 2
only a small fraction of the antibody free in solution. This antibody is unmasked during
affinity purification and becomes detectable by ELISA.
It is possible that limited denaturation of the antibodies occurs during purification,
altering their monospecificity. For example, it has been demonstrated that the binding site
of M11, a murine, monoclonal antibody, is altered after purification at a low pH (2.2),
resulting in its binding to many different proteins (McMahon and O’Kennedy, 2000). The
inference is that such a polyreactivity represents an acquired, artificial characteristic.
Because chemical fixation prevents cellular disintegration, it is the foundation for almost all
microscopic studies. It is the most widely used method for preserving specimens for light and
electron microscopy. The reasons for its universal use are that it adequately preserves many
cellular components, including soluble and structural proteins; prevents autolysis and dis-
placement of cell constituents, including antigens and enzymes; stabilizes cellular materials
against deleterious effects of subsequent preparatory treatments, including incubations; and
facilitates conventional staining and immunostaining. Fixation also allows the clarity of struc-
tural details shown by photomicrographs and electron micrographs, and it can be applied eas-
ily. No other tissue preservation method can claim these advantages. Freezing is a useful
adjunct to chemical fixation. The biochemical reactions of formaldehyde and glutaraldehyde
with proteins are discussed here and in more detail elsewhere (Hayat, 1986, 2000a).
An understanding of the effects of fixation on antigens and cell morphology is a pre-
requisite to accepting the validity of immunohistochemistry. It is necessary to know the
extent of antigen preservation or destruction due to fixation and dehydration-embedding.
The preservation of antigenicity is adversely affected by any type of chemical fixation
because epitopes may be masked sterically by the surrounding soluble proteins in the tis-
sue fluid. The fixative may crosslink another molecule (usually a protein) directly to the
epitope or in the vicinity of the antigen. The former may allosterically alter the epitope
configuration, inhibiting its recognition by the antibody. The latter may block the accessi-
bility of the antibody to the antigen. In addition, the antigen may be degenerated by direct
crosslinking with the aldehyde groups of the fixative. All of these changes may be
reversible or irreversible or partly reversible or irreversible.
FORMALDEHYDE
53
54 Chapter 3
Aqueous formaldehyde as used for fixation contains mostly methylene glycol (~99%),
its oligomers, and small amounts of formaldehyde. The proportion of the oligomers present
depends inversely on the temperature. Formaldehyde solution cannot be obtained without
the formation of methylene glycol. It is not the formaldehyde molecule that is primarily
responsible for rapid penetration into the tissue but methylene glycol, which is the major
component of formaldehyde solution. At concentrations of 2% or less, the formaldehyde in
solution is present practically only as the hydrated monomer
Methylene glycol is formed by the reaction between formaldehyde and water:
has an available hydrogen site, a reactive hydroxymethyl group is produced, which is the
addition product. Subsequently, a second hydrogen site may react with this addition prod-
uct, yielding a methylene bridge between two amino acids in the protein. As a result of this
interaction a large number of hydroxymethyl groups are produced, utilizing reactive
species on the side chains of amino acids or directly from the peptide bond (Table 3.1). In
other words, additional reactions of formaldehyde with the hydroxymethyl groups and pre-
viously unreacted amino acid side chains form the methylene bridges that are the protein
crosslinks of formaldehyde fixation. Formaldehyde reacts through several steps to form a
methylene bridge between two neighboring amino groups of amino acids. Neutral pH
favors the formation of such bridges.
The primary reason for using neutral-buffered formalin is that at this pH hydrogen
sites in peptide molecules are available for linkage because they are in an uncharged state.
In contrast, an acid pH induces formation of charged amino groups that lack reactive
hydrogen sites. Nonscientific reasons for using formalin are that it is inexpensive, and eas-
ily available, and diagnostic pathologists and technicians have been trained to use it.
Formaldehyde introduces both intramolecular and intermolecular crosslinks between
proteins involving hydroxymethylene bridges, which change the three-dimensional struc-
ture of proteins. Such changes involve the tertiary and quaternary structures of proteins,
whereas the primary and secondary structures are little affected. It has been shown that the
secondary structure of purified protein molecules remains mostly unaltered during fixation
with formaldehyde (Mason and O’Leary, 1991). Even when the quaternary structure is
changed by formaldehyde fixation, the secondary structure can remain intact.
Formaldehyde does not react with all functional groups in proteins with equal rapid-
ity; it reacts first with lysine and cysteine, and subsequently these amino acids are
56 Chapter 3
crosslinked to glutamine, asparagine, and arginine. This does not mean that the fixative
alters the amino acid sequence of the antigen molecule; the structure of the epitope is
maintained even though it may be masked. However, various proteins respond differently
to fixation with formaldehyde. It is relevant to point out that aldehyde fixation does not
completely abolish the selective permeability of cell membranes (Hayat, 1986). Therefore,
it can be inferred that membrane proteins and other proteins are not destroyed by fixation
with formaldehyde.
The strongest indirect evidence in support of the role of protein crosslinking in creat-
ing a barrier to the penetration of antibodies and their reaching to the epitopes emerges
from the crosslinking ability of glutaraldehyde. It is known that this dialdehyde introduces
extensive protein crosslinks that are mostly irreversible, which significantly inhibits anti-
genicity (Hayat, 1986, 2000a). This inhibition is due to the crosslinking of protein antigens
as well as crosslinking and compacting of proteins surrounding the antigen molecule. Most
epitopes in most protein antigens studied so far are located at or near the exposed surface
of the antigen molecule. Therefore, compacted proteins surrounding the antigen molecule
will prevent the recognition of epitopes by the antibody. The small size of the epitopes
makes them susceptible to masking by the surrounding crosslinked proteins.
The presence of the two aldehyde groups is the reason for its being the most effective
protein crosslinking molecule. Glutaraldehyde introduces both intramolecular and intermol-
ecular protein crosslinks that are predominantly irreversible during subsequent processing,
including incubations of the tissue. This versatile property results in excellent preservation
of the ultrastructure; however, it also has limitations. The dialdehyde produces strong, irre-
versible protein crosslinks when used in standard concentrations (2–3%). Therefore, it
masks most epitopes. In addition and more important, steric hindrance resulting from
extensive crosslinking of cellular proteins in general inhibits the antibodies from reaching
the intracellular epitopes. Thus, glutaraldehyde is less suitable for light and electron micro-
scopic immunocytochemistry. Some hardy antigens that are less susceptible to the effect of
aldehydes, however, can be studied following fixation with glutaraldehyde alone or in com-
bination with formaldehyde for electron microscopic immunocytochemistry.
For light microscopic immunohistochemistry and immunocytochemistry, formaldehyde
is preferred over glutaraldehyde because it is a monoaldehyde and thus penetrates the
tissue more quickly but crosslinks proteins more slowly than glutaraldehyde, a dialdehyde.
Formaldehyde does not react with all the functional groups in proteins with equal rapidity.
In its reactions with proteins, the first step involves the free amino groups with the formation
of amino methylol groups, which then condense with other functional groups such as
phenol, imidazole, and indole to form methylene bridges The occurrence of these
bridges is considered responsible for the fixation of proteins by formaldehyde under
conditions of fixation.
The process of masking is progressive, i.e., the longer the fixation, the stronger the
crosslinking as well as epitope masking. It is well established that with increasing dura-
tions of fixation, antibodies show a nearly linear decrease in immunostaining, indicating a
successive masking of epitopes (Werner et al., 1996). Therefore, the duration of fixation
should be taken into account while determining the duration of treatment for epitope
retrieval. Generally, mild fixation with formaldehyde is preferred. Some epitope types are
not affected by standard or prolonged fixation with formaldehyde or a mixture of
formaldehyde and glutaraldehyde or, as stated earlier, glutaraldehyde alone.
The advantage of using a mixture of formaldehyde (4%) and glutaraldehyde (~0.1 % or
a lower concentration) is improved preservation of cell structure. Although formaldehyde is
better than many other fixatives for preserving morphological details, it is far inferior to glu-
taraldehyde. The quality of morphological preservation with glutaraldehyde has been com-
pared with that obtained with formaldehyde (Hayat, 2000a). These studies clearly indicate
superior structural preservation with glutaraldehyde. In fact, fixation with formaldehyde is
unacceptable for routine electron microscopy, which demands superior structural preserva-
tion because of its high resolving power. However, for most electron microscopic immuno-
cytochemical studies, a mixture of formaldehyde and glutaraldehyde is recommended
because many types of epitopes are irreversibly masked when the latter alone is used.
Although at present formalin is routinely used for the fixation of surgical tissue specimens,
the advantage of a mixture of formaldehyde and glutaraldehyde cannot be overemphasized.
There is no optimal universal fixative for all types of antigens, so the choice of a fix-
ative depends on the type of epitope and the tissue under study. Furthermore, the selection
58 Chapter 3
of a fixative is always a compromise between the preservation of antigenicity and cell mor-
phology. Although the best fixative for a specific epitope is determined by trial and error
or by recommendations from a published study, 4% formaldehyde in 0.1 M buffer (pH 7.2)
is recommended for adequately preserving both the antigenicity and cell morphology for
immunostaining. Alternatively, 10% neutral-buffered formalin can be used. Standard fixa-
tion for 4–6 hr at room temperature is considered a mild and effective fixation.
Most of the reactions of this aldehyde with proteins during mild fixation are
reversible, and the resultant protein crosslinks for the most part can be weakened and/or
broken with treatments such as heating. Unbound formaldehyde is also removed during
washing. The reactions of formaldehyde with proteins are influenced by several factors,
including its concentration, pH, temperature, and duration of fixation. In general, higher
values of these parameters result in increased binding of formaldehyde. The maximum
binding occurs at pH 7.5 to 8.0.
However, it should be noted that the fixation throughout the tissue block is completed
in ~24 hr, but pathological specimens are usually fixed for less than this duration before
their further processing. As a result, the tissue is only partially fixed with the aldehyde, and
its fixation is completed with the dehydrating ethanol. It means that the tissue is fixed
partly by protein crosslinking with the former and partly by coagulation with the latter
(Battifora, 1991). Consequently, the specimen may show heterogenicity of immunoreac-
tivity; different areas of the section may exhibit different intensity of staining. Variable
staining density in different areas of the tissue block may also be caused by the presence
of air bubbles in the vial containing the specimens during fixation. Tissues fixed with coag-
ulating fixatives such as ethanol generally do not benefit by antigen retrieval treatments.
Although 10% neutral buffered formalin is the most commonly used fixative and
yields satisfactory results, the optimal preservation of certain antigens requires a different
fixative. Three examples are given. The immunostaining of transforming growth
and thrombomodulin in the human skin has been reported to be superior in the specimens
fixed with methanol-Carnoy’s solution (methanol: chloroform: acetic acid, 6:3:1) com-
pared with that in the formalin-fixed specimens (James and Hauer-Jensen, 1999). Optimal
duration of fixation is 24 hr. Similarly, the antigenicity of the proteinase-K–resistant form
of the prion protein in brain tissue is better preserved in the Carnoy-fixed specimens
(Giaccone et al., 2000). Another example is leukocyte antigenicity in various rat tissues,
which is better preserved in the Carnoy-fixed specimens (Shetye et al., 1996).
conformation of the antigen molecule but also create a barrier to antibody access to the
epitope. The formation of extensive crosslinkages during prolonged fixation for weeks or
months is supported by the observation that such tissue blocks become hard and difficult
to section. It is well known that archival tissues are difficult to section.
The effect of prolonged fixation with formaldehyde on the antigenicity of the nucleus
may differ from that of the cytoplasm. This phenomenon is exemplified by Bcl-2 and Bax,
members of the same family of proteins involved in apoptosis regulation; these proteins
reside in the cytoplasm as well as in the nucleus. It was recently demonstrated that pro-
longed fixation with formaldehyde alone irreversibly reduced nuclear or mitotic Bcl-2
immunoreactivity even after heat-mediated antigen retrieval in monolayers of MCF-7
human breast cancer cells (Hoetelmans et al., 2001).
Heat treatment, on the other hand, elevated cytoplasmic immunoreactivity of Bcl-2.
However, nuclear and mitotic Bcl-2 immunoreactivity was clearly present when these cells
were fixed with formaldehyde (3.6%), followed by postfixation with methanol for 10 min
at –20°C. Treatment with ice-cold methanol makes the cell membrane permeable, allow-
ing antibody access to intranuclear antigens without protein relocalization. Extensive pro-
tein crosslinking with formaldehyde is required for maintenance of intranuclear Bcl-2
immunoreactivity. In contrast to Bcl-2, Bax immunoreactivity was detected in nuclear and
cytoplasmic compartments regardless of the duration of formaldehyde fixation used.
In light of the aforementioned information, when tissue specimens are exposed to
formaldehyde for longer durations due to unavoidable circumstances, immunohistochem-
ical findings should be interpreted with caution, as many tissue antigens could be lost or
irreversibly masked.
Another fixative, Kryofix (E. Merck, Darmstadt, Germany) has been recommended
as a replacement for formaldehyde in immunohistochemistry by Boon and Kok (1994).
Kryofix is a coagulant fixative containing 50% ethyl alcohol and polyethylene glycol (mol.
wt. 300). Both ethyl alcohol and polyethylene glycol diffuse rapidly into the tissue, and the
tissue fixation is completed in Kryofix in 90 sec with microwave heating. I do not have
personal experience with Kryofix.
Fixation Conditions
Some major factors that adversely affect immunostaining are delays in transferring
the tissue blocks into the fixative after their surgical removal as well as shorter or longer
than optimal duration of fixation. Any delay in the exposure of the tissue to the fixative
invites increasing proteolytic degradation of antigenicity. Therefore, if possible, surgical
tissues must be placed directly into the fixative immediately after their removal. If delay is
unavoidable, the tissue can be refrigerated prior to fixation.
It is not uncommon for thick (~5 mm) surgical tissues excised for diagnostic pathol-
ogy to be underfixed with formalin, especially the core of the tissue block. As an average,
fixation in formalin solution for less than 24–48 hr, depending on the size of the tissue
block, tends to crosslink only the periphery of the specimen. Under this condition, the core
of the tissue block either remains unfixed or fixed by coagulation with the alcohol used
subsequently for dehydration. Sections cut from the core tend to show autolysis and inad-
equate immunostaining, resulting in false-negative staining. Such inadequate staining can
be improved by attaching paraffin sections to glass slides and then removing the paraffin
with an organic solvent (Eltoum et al., 2001). This treatment is followed by rehydration,
buffer rinse, and refixation with formalin. The fixative is removed by rinsing with buffer
before staining. If necessary, incompletely fixed, paraffin-embedded tissues can be
deparaffinized and refixed and reembedded, risking some damage to cell morphology and
immunogenicity. These corrective methods become useful only when additional tissue
specimens are not available.
Overfixation of specimens may also be encountered, resulting in weak or absent
immunostaining, depending on the susceptibility of a specific antigen to the fixative. As
already emphasized, prolonged fixation introduces excessive protein crosslinking, which
hampers antigen accessibility to the antibodies. In addition, 10% formalin solution contains
only 4% formaldehyde, and the remaining components may damage antigens during pro-
longed fixation. If prolonged fixation has been carried out, immunostaining can be increased
by the following steps: robust antigen retrieval by heating, higher antibody concentrations,
longer durations of incubations in the reagents, and signal amplification. To determine the
optimal increase in heat or protease treatment to counteract the effects of prolonged fixation,
three slides can be processed with progressive doubling of the duration of treatment (Werner
et al., 2000). The best stained slide of the three is used for interpreting the results of the study.
However, some of these approaches may increase the background noise.
Overfixation of specimens is also recognized by difficulty in sectioning because of
excessive hardness of the tissue. This problem arises when tissues are fixed with formula-
tions containing ethanol, methanol, or acetone. Excessive dehydration with an organic sol-
vent may also cause tissue hardness, especially of small specimens (1–2 mm). Such
Fixation and Embedding 61
specimens may shatter instead of being sliced during cutting. The increased hardness can
be prevented by shortening the duration of dehydration. If the tissue has been excessively
hardened, it can be partially corrected by briefly soaking in water (Eltoum et al., 2001).
Water tends to penetrate up to 0.5 mm into the tissue block. A large tissue block can be cut
into small pieces before soaking in water. Overfixation with protein crosslinking aldehy-
des can also be partially reversed with washing in water or aqueous buffers.
and cheaper and can be completed in a conventional microwave oven with a maximal
power output of 650 W or higher. A water load (200 ml in a beaker), placed in a rear cor-
ner of the oven, serves as a damper to increase heating times to 10 sec or longer. Tissue
specimens (e.g., nervous, ocular, and skin tissues) are fixed for 2 min at room temperature
with 2% in 0.2 M sodium cacodylate buffer (pH 7.2, 450 mOsm). They are trans-
ferred into small glass vials containing 10 ml of the fixative. The vials are placed in
the center of the microwave oven and heated at maximal power output and at a frequency
of 2,450 MHz until the temperature between 43°C and 40°C is attained (~12 sec). The
specimens are removed from the oven and kept at room temperature for 10 min prior to
washing in 0.1 M buffer and embedding in an epoxy resin. The results of this procedure
are shown in Fig. 3.1.
64 Chapter 3
Typically, residual enzyme activity is localized at the subcellular level after fixation
with an aldehyde followed by incubations. Aldehydes, especially glutaraldehyde, denature
enzyme molecules to various degrees. Lengthy incubations under nonphysiological condi-
tions may cause the loss of structural details. To improve preservation of both the enzy-
matic activity and the ultrastructure, incubation durations can be shortened under
microwave heating (Rassner et al., 1997). The advantage of incubation under microwave
heating becomes apparent when one considers that it allows incubation of tissue
specimens, whereas conventional incubation requires tissue slices of 0.1 mm.
Tissue specimens are fixed for 16 hr with a mixture of 2% paraformaldehyde
and 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) containing 0.06% calcium
chloride. After rinsing in buffer, the specimens are placed in 10-ml snap-cap glass vials
containing 2 ml of incubation medium. The vials are capped to avoid contamination of
the incubation medium from the water bath. They are placed into a staining jar (3 × 3.5 ×
2.5 inches) which has been filled with 150 ml of tap water at 22°C. The microwave incu-
bations are carried out twice for 30 sec each in a microwave oven at the highest power set-
ting (900 W at 2,450 MHz, with two water loads 250 ml each) placed in the rear of the
oven. The temperature in the oven is not allowed to exceed 40°C.
The water of the water bath is changed between the two 30-sec pulses. The tempera-
ture of the water bath rises during microwave heating from 22°C to 40°C. The staining
jar is removed from the oven, and the incubation is continued for an additional 30 min at
37°C. After washing with distilled water, the specimens are postfixed with a mixture of 1%
and 1.5% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.2) for 90 min in
the dark at room temperature. The specimens are rinsed in buffer and then immersed in 2%
aqueous uranyl acetate. Detailed methods for achieving accelerated visualization of acetyl-
cholinesterase activity at motor endplates using microwave heating have been presented by
Petrali and Mills (2001).
The activity of some enzymes can be preserved by tissue fixation with aldehydes in
a microwave oven at a low temperature for electron microscopy. This procedure has been
used for observing cytochrome oxidase in mitochondria in hamster submandibular gland
tissue (Moriguchi et al., 1999). The activity of this enzyme has also been studied in the iso-
lated mitochondria of this tissue, using the same method (Moriguchi et al., 1998). This
procedure also has been employed for confocal laser scanning microscopy of enzymatic
activity (Moriguchi et al., 1999). The following protocol was used for processing the tis-
sue for electron microscopy.
The microwave processor is fitted with a thermometer, timer, stirrer, and power level
controller (150 W to 400 W) (MI-77, Azumaya Company, Tokyo, Japan). One glass Petri
dish with 70-ml capacity containing 50 ml of a mixture of 2% paraformaldehyde and 0.5%
glutaraldehyde containing 45 mg/ml sucrose in 0.1 M PBS (pH 7.4) is placed in a large
Petri dish containing 250 ml of chilled water in the processor. The specimens are heated in
a microwave oven for 10 min at ~4°C at a low energy level of 150 W.
Fixation and Embedding 65
Cryoinjury to the specimen is caused directly by extra- or intracellular ice crystal for-
mation as well as by ice-induced solution effects during cryopreservation. Ice crystals seri-
ously deform cell components. Another disadvantage of the formation of ice crystals near
the specimen surface is slowing the cooling rate in areas below the surface because their
thermal conductivity is about half that of solid water in a noncrystalline state. Furthermore,
ice crystal formation is accompanied by the generation of latent heat, which also slows
down the freezing rate.
Apparently, cryoinjury can be avoided by eliminating ice crystal formation and vitrify-
ing the specimen. Vitreous state can be achieved by ultrarapid cooling (> ) or using
high concentrations of a cryoprotectant. However, the former is difficult to attain, and the lat-
ter tends to cause chemical toxicity and high osmotic stress. Because biological specimens
possess low thermal conductivity and high thermal capacity, ultrarapid cooling can be
obtained only for very small specimens. The above-mentioned difficulties in obtaining ultra-
rapid freezing can be minimized in the presence of microwave heating. This treatment sup-
presses ice crystal formation near the specimen surface, thereby extending the depth of good
freezing from the specimen surface. Another advantage is better reproducibility of results
because the state of water near the specimen surface is under control with microwave heating.
Two mechanisms responsible for decreased rate of ice crystal growth are suggested.
It is possible that the electric field component of electromagnetic radiation interacts with
dipolar water molecules, disrupting the ice nucleation phenomenon (Hanyu et al., 1992).
In other words, microwave heating reduces the size and number of ice crystal nucleation
centers near the specimen surface. An alternative explanation is based on microwave radi-
ation interfering with the kinetic processes of ice crystal growth (Jackson et al., 1997). For
an ice crystal to form and grow, each water molecule must have an appropriate spatial ori-
entation, position, and energy. Rapid ice crystal growth requires the molecular clusters to
share edges and faces with the ice lattice without the induction of mutual strains. The
torques produced by a microwave field can increase the number of available isomeric con-
figurations, reducing the likelihood of a cluster of molecules having a configuration suited
to integrating into a crystal lattice. Further development in the application of microwave
heating to vitrification of biological specimens is awaited.
Two apparatuses have been constructed for achieving ultrarapid freezing in the pres-
ence of microwave heating (Hanyu et al., 1992; Jackson et al., 1997). Microwave treatment
can be employed with or without a cryoprotectant. According to one method, microwave
heating is used at 2.45 GHz for a short duration (50 msec) immediately before and during
tissue contact with the surface of a copper block cooled with liquid nitrogen (Hanyu et al.,
1992). The ultrastructure is well preserved to a depth of from the contact surface,
which is comparable to the depth obtained by the metal contact method using liquid
helium in the absence of microwave heating.
PARAFFIN EMBEDDING
For routine immunohistochemestry, surgical and other tissues are embedded in paraf-
fin which is a mixture of hydrocarbons. Automated paraffin tissue processors are com-
mercially available that customize the schedule to meet specific needs. However, tissues of
66 Chapter 3
various types and sizes as well as the particular study objective require optimal conditions
of dehydration, infiltration, and embedding in paraffin. Chemical and physical changes
occur in specimens during these treatments, affecting the sectioning and immunostaining
qualities. Longer than optimal durations of these steps is a common habit, resulting in hard
and brittle tissues that are difficult to section. It should be noted that additional fixation
occurs during dehydration, accompanied by antigen masking and lipid dissolution.
After fixation, a series of ethyl alcohol (a water-miscible solvent) of ascending con-
centrations is used to remove water from the tissue. Free water from the tissue is easily
removed by diffusion. Water attached to the tissue by hydrogen bonds is also replaced by
ethyl alcohol of higher concentrations. The efficacy of a solvent depends on its hydrogen
bonding strength and molecular weight (Wynnchuk, 1993). Higher temperatures, vacuum,
and microwave heating expedite the speed of dehydration, allowing shorter durations of
dehydration.
Xylene (an aromatic hydrocarbon) is used to replace ethyl alcohol from the tissue
before infiltration with paraffin. Xylene is miscible with ethyl alcohol and paraffin. Xylene
is called a clearing agent because it has a refractive index similar to that of proteins and
thus renders tissue more or less transparent. It is generally satisfactory when the tissue
blocks are not thicker than 3–4 mm. Xylene must be completely removed with paraffin,
otherwise tissue will not section. Excessive exposure to xylene causes further denaturation
of tissue proteins, causing difficulties in sectioning. Some lipid extraction also occurs in
the presence of xylene. The treatments mentioned above to expedite the diffusion of ethyl
alcohol also speed up the penetration of xylene. Xylene is also used between ethyl alcohol
and mounting sections with resinous mounting medium after staining. The volatility and
inflammability of xylene render it potentially dangerous. It must be used in a fumehood.
While tissue is in xylene, gradual infiltration with paraffin is carried out. For tissues
of a small size, 2 to 3 hr of paraffin infiltration is adequate. For large tissues (5–10 mm),
overnight infiltration is required. The temperature during infiltration must not be higher
than 4° above the melting point of paraffin (54–58°C).
Vacuum embedding can be carried out to remove air bubbles from the tissue and rap-
idly replace the clearing agent with paraffin. This approach is especially desirable for air-
containing tissues such as lung or hard tissues such as fibrous or scar tissues. The vacuum
should not exceed 400–500 mm of mercury to avoid damage to the tissue.
Paraffin blocks are trimmed with a scalpel, a razor blade, or a hot spatula and
mounted on wooden or fiber blocks. While being sectioned, longitudinal block edges must
be parallel to the knife edge to obtain a ribbon. Paraffin sections of any thickness show
compression, which is usually relieved when they are floated on a glass slide and dried.
It should be noted that the melting point and crystalline structure of paraffin influence
the section quality. Paraffin of a lower melting point is less brittle when solidified; how-
ever, it tends to show more compression during sectioning. On the other hand, paraffin of
a higher melting point provides a better support for hard specimens. Paraffin of a smaller
crystalline structure adheres closely to the cell components in the embedded tissue, pro-
viding good support for sectioning. Paraffin sections of a larger crystalline structure show
more pronounced curvature, which is difficult to flatten after sectioning. To obtain crystals
of a small size, paraffin should be cooled rapidly. Deeper layers of the tissue block contain
larger and looser crystals, resulting in poor quality of sections in these layers.
Fixation and Embedding 67
Vacuum combined with microwaving has been tried for embedding the tissue in
paraffin, using Milestone’s MicroMED LAVIS-1000 machine (Marani et al., 1996; Bosch
et al., 1996). The advantage of this system is that microwaves travel with ease through a
vacuum, whereas conventional heating under vacuum is difficult. This machine provides
pressure reaching 100 hPa, and the microwave oven attains a maximum power of 1,000 W;
its cycle time can be adjusted between 0.1 and 0.5 sec. The machine is equipped with an
infrared temperature probe which allows temperature control from outside the unit.
To obtain satisfactory results, coordination of temperature with vacuum is necessary.
Paraffin embedding is carried out in a stepwise descending series: 700 hPa, 500 hPa,
300 hPa, and 100 hPa. A too-rapid lowering of the pressure is damaging to tissue mor-
phology. The temperature during dehydration with isopropanol should not exceed 60°C.
Further improvements of this system are awaited.
Two types of forces are exerted during paraffin sectioning: flow shearing and point-
to-point shearing (Allison, 1998). Flow shearing proceeds ahead of the cutting edge,
resulting in smooth sections. In contrast, point-to-point shearing travels through the path
of least resistance ahead of the cutting edge, producing a section of uneven thickness.
Paraffin contains additives that minimize the point-to-point shearing and reduce the
plastic flow. Additives are synthetic polymers that improve the consistency of paraffin by
filling the spaces among paraffin crystals in the tissue.
Cut a paraffin block containing one tissue specimen with a razor blade,
and mount it to a support stub. Trim the block manually with a razor blade or on an auto-
matic trimming microtome to a rectangular or trapezoidal cutting face. The size of the
block face is determined by the objective of the study and the size of the tissue specimen.
The upper and lower edges of the block facing the knife cutting edge should be parallel to
each other to obtain a ribbon, if required. Mount and orient the block on the microtome,
so that its longer edge is parallel to the cutting edge. A steel or glass knife can be used.
Cut sections ( thick) on a rotary microtome, which usually has an automatic
advance mechanism that can be set to advance the specimen block the desired distance
toward the knife with each stroke. Manual or motorized rotary microtomes are commer-
cially available (Triangle Biomedical Sciences, Durham, NC; Sakura Finetek, Torrance,
CA). A microtome with automated specimen approach, trimming, and sectioning is also
available (Leica Microsystems, Deerfield, IL). Float the sections on water or 4% formalin
on a glass slide and heat for 10–15 min at ~40°C on a warming plate to remove the com-
pression. Remove the liquid with a fine pipette and dry overnight in an oven at ~40°C to
ensure section adherence to the slide. Drying can also be accomplished in 15 min at
~40°C in a microwave oven. Note: Sectioning will be adversely affected if tissue infiltra-
tion with paraffin is incomplete. A too-shallow or too-steep bevel angle of the steel knife
relative to the tissue block face will result in section compression and chatter, respectively.
The optimal cutting angle is 4°. Section adhesion to the glass slide can be ensured
by coating the slide with polylysine ( to 1 mg/ml) in 10 mM Tris (pH 8.0).
Alternatively, coated slides are commercially available (Probe-On-Plus slides from Fisher
Scientific).
Make sure that the tissue specimen is firmly mounted to the stub and the latter to the
microtome. Also, the paraffin block should be fairly cold at the time of sectioning. Low
humidity in the vicinity of the microtome tends to result in static electricity, which makes
it difficult to separate the section from the block face after cutting. For additional details,
see Ruzin (1999).
A vacuum–microwave combination has been used for processing tissues for light
microscopy (Kok and Boon, 1996), transmission electron microscopy of animal tissues
(Giberson, 2001) and botanical specimens (Russin and Trivett, 2001), and scanning elec-
tron microscopy of human lymphocytes (Demaree, 2001).
The vacuum-microwave heating method is especially useful for processing botanical
tissues because these specimens possess physical characteristics that hamper easy pene-
tration of reagents; these characteristics include cell wall, vacuoles, plastids, and intercel-
lular spaces. Secondary cell walls may contain cutin, suberin, and lignin, which are
hydrophobic. These waxy substances limit the evaporation of water from the tissue and
resist the penetration of reagents. The presence of air in the intercellular spaces creates a
barrier to fixative penetration. These impediments to reagent penetration and action of fix-
atives can be significantly reduced by using vacuum–microwave heating. For details of this
methodology, see Russin and Trivett (2001).
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Chapter 4
Many factors influence antigen retrieval, including fixation, heating, retrieval fluid, and
antibodies.
FIXATION
Fixation is the most important factor affecting antigen retrieval. Type of fixative and
duration and temperature of fixation are all important. Many varieties of epitopes have
been retrieved with various degrees of success in tissues fixed with formalin, methanol,
methacarn, or Bouin’s fixative; buffered 10% formalin containing 3.7–4.0% formaldehyde
is the most commonly used. Although fixation with paraformaldehyde or glutaraldehyde
better preserves cell morphology because of stronger, and more rapid and more extensive
protein crosslinking, antigen unmasking becomes difficult.
The use of formalin has become a matter of habit and convenience, especially in
pathology laboratories; it is also inexpensive. To improve the preservation of cell mor-
phology, it is recommended that a mixture of formalin (or paraformaldehyde) and
glutaraldehyde (0.05–0.5%) be tried. Such mixtures are routinely employed for immuno-
cytochemical studies with the electron microscope. It is known that some types of antigens
are resistant to fixation with low concentrations of glutaraldehyde. Preembedding immu-
nocytochemistry by the avidin-biotin method, which avoids fixative effects, has been suc-
cessfully applied for identifying peptide or protein antigens in the brain tissue fixed with
glutaraldehyde (Mrini et al., 1995).
The effect of fixation on antigenicity is complex. With the exception of a minority of
antigen types (e.g., PCNA nuclear protein) that are formalin resistant to various degrees,
most antigens are sensitive to the concentration of the fixative and the duration of fixation.
Although 10% formalin is the usual fixative, it is inadequate for preserving some types of
antigens that are fixative resistant. It has been demonstrated histochemically, for example,
that PCNA nuclear protein antigenicity is preserved much better with 20%
formalin-PBS than with 10% of the same fixative (Muñoz de Toro de Luque and Luque,
1995). The reason is that the protein antigen is partially extracted with the lower fixative
71
72 Chapter 4
concentration. In this respect, it should be noted that some types of antigenicity, such as
PCNA, require quantitative measurements for assessment of clinical significance, as its
low levels may be present in quiescent cells.
Is antigenicity affected by factors other than fixation? Yes. Although fixation is the most
important factor in tissue processing for immunohistochemistry, other factors tend to affect
immunorecognition of antigens. These factors include the interval between removal of the
tissue from the human or animal and fixation; the method of excising the tissue from the
body (mechanical damage); the technique of cutting sections of the tissue embedded in
paraffin or resin; the procedures for removing section compression, folds, or bubbles and
attaching it to the glass slide; the interval between cutting sections and immunostaining (stor-
age or without storage of slides prior to staining); and other immunostaining details. Folds
and bubbles hinder section adhesion to the slide, and they may also show 3,3-diaminoben-
zidinetetrachloride (DAB) precipitation. Bubbles under sections may appear as brown spots
on immunostained sections (Grizzle et al., 2001). Paraffin sections ( thick) adhere tena-
ciously to glass slides by heating overnight at 65°C. The use of a PAP pen or other means to
demarcate the tissue to aid in staining is also a variable. Awareness of the above-mentioned
variables should prevent erroneously attributing them to problems with fixation.
Tissue specimens ideally should be placed in the fixative immediately after their
removal from the body. This problem arises in studies of human tissues, for their immedi-
ate fixation is usually not feasible. If immediate fixation is not possible, the tissue must be
kept cool and moist by covering it with a piece of cloth soaked in sterile, cold saline for
not more than 20–30 min. During this time the specimen should not contact any dry and
absorbent object such as paper, a paper towel, or gauze. To keep the paper trail of the spec-
imen (source, time, place of collection, etc.) is no less important.
Note that even human tissues fixed immediately after their removal from the body
may undergo cellular changes because usually the vascular supply is terminated before the
tissue is surgically removed. During this duration (~1 hr) the tissue remains at body tem-
perature, at which the activity of digestive enzymes continues, damaging the cellular struc-
tures (Grizzle et al., 2001).
Chemical fixation is not the only factor that causes loss or irreversible masking of anti-
gens. Treatments such as dehydration and embedding following fixation also play a role in
the loss of immunorecognition of antigens. Absolute ethanol and xylene must not contain
traces of water, and the water bath should be very free from contaminants such as bacteria,
fungi, dust, and dirt. Once the sections are contaminated, they cannot be decontaminated.
According to Watanabe et al. (1996), the antigen preservation test (Riederer, 1989)
showed that immunostaining intensity, for example of decreased during fix-
ation with paraformaldehyde but did not decrease during washing and immunostaining.
The proportionate decrease in intensity due to fixation was almost constant even when the
amount of the antigen differed in the sections. They concluded that the decrease in
immunostaining intensity was related to a proportional decrease in antibody binding due
to masking of antigens during fixation.
DENATURATION
On the basis of antigen retrieval obtained with protein denaturing agents, it has been
proposed that in certain cases antibodies recognize denatured but not native antigens.
Factors Affecting Antigen Retrieval 73
However, this proposition seems untenable, given the requirement of a specific amino acid
sequence for an epitope, as well as a specific conformation of antibody molecule, in order
for antigen-antibody binding to occur. How could an antibody react with a completely
denatured antigen, when the former is usually generated using the native form of the
latter? For example, antibodies generated against selected regions (N-terminal fragment or
C-terminal region) of corresponding antigens recognize predominantly similar undena-
tured regions, unless these regions of the antigen are masked by other regions of the
antigen and/or by some surrounding components.
The proposition that antibodies recognize certain antigens only after the latter have
been denatured is true only when the epitope is unmasked and remains undenatured fol-
lowing antigen denaturation. In other words, a denatured epitope cannot be recognized by
the antibody if the amino acid composition of the epitope peptide and/or its linear amino
acid sequence is altered or damaged. The reaction between the antigen and the antibody is
dependent on the conformation of the former. However, the presence of an intact three-
dimensional folded antigen structure may not be necessary in certain cases for antibody
binding. Denaturation or unfolding of certain antigen molecules may be necessary to
unmask the epitope that is buried in the interior of the folded antigen structure (personal
communication, Dennis Brown).
It is likely that most antigen molecules form multiprotein complexes, resulting in
masking of epitopes by surrounding proteins. The epitope masking becomes more serious
when these complexes are crosslinked with formaldehyde. Such masked epitopes can be
recognized by the antibody only when exposed by breaking crosslinks and denaturing
surrounding cell components. If this is so, denaturing treatments cause breakdown of
reversible crosslinks introduced by formaldehyde and denaturation of surrounding cell
components, enabling the antibodies to recognize the native, uncrosslinked or partially
crosslinked undenatured structure of the reactive epitope molecule. It means that denatur-
ing treatments do not denature antigen per se but denature multiprotein complexes of
which the antigen is a part. It may be that imprecise nomenclature has given rise to con-
fusion in this field.
It should also be noted that denaturing agents make cells permeable, facilitating anti-
body penetration. Moreover, these agents are used usually in combination with microwave
heating. Therefore, the role of these agents in epitope retrieval needs to be explained in
the context of their role in cell permeabilization as well as of the influence of elevated
temperatures.
HEATING
pH
Another important factor in achieving optimal antigen retrieval is the pH of the retrieval
solution. It is thought that the pH is more important than the constituents of the retrieval fluid
(Shi et al., 1995a). There is, however, no universally optimal pH for a retrieval fluid. The
retrieval of most types of antigens requires a specific pH, although retrieval of a few anti-
gens can be achieved over a wide range of pH levels; for example, AE1 and NSE (cyto-
plasmic antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens) can
be retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, following
retrieval at pH 3–6, some antigens (e.g., estrogen receptor) display a marked decrease
in the immunostaining, while still other antigens such as cytoplasmic HMB 45 show weak
or negative staining after retrieval at pH 1–2 but excellent results in the high pH range
(Shi et al., 1995a).
Some antigens can be retrieved only at a low pH. These types are exemplified by
thrombospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and
2.5, respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). However, pH levels
lower than 3.0 can severely damage tissue morphology. Low pH levels can also alter the
localization of some cytoplasmic antigens, resulting in false-positive staining of the
nucleus. For example, an antibody (UCHL1) to T cell antigen is effective at pH 6.0 but
results in the staining of every nucleus at pH 2.0 (personal communication, H.Y. Lan).
In summary, the use of sodium citrate buffer at pH 6.0 increases the intensity and
extent of immunostaining of a wide variety of tissue antigens, whereas Tris-HCl buffer
may yield better results for some antigens at pH 10.0. On the other hand, low-pH antigen
retrieval fluids are necessary for some antigens such as thrombospondin. For previously
unexamined antigens, a test battery based on three pH values (low, middle, and high)
should be carried out to establish an optimal protocol (Shi et al., 1996a).
MOLARITY
The concentration of antigen retrieval fluid is often less important than temperature,
duration of heating, and pH in achieving optimal antigen retrieval. For example, sodium
citrate buffer is effective at molarities ranging from 0.01 to 0.5. However, in the case of
another antigen retrieval solution, ammonium chloride, 0.5%, 1%, 2%, and 4% solutions
were tested, and 4% concentration yielded the best immunostaining of vimentin in
archival paraffin sections (Suurmeijer and Boon, 1993a). Ammonium chloride solutions
are weakly acidic (pH 3–4). According to Bruno et al. (1992) and Muñoz de Toro de
Luque and Luque (1995) minor changes in ionic strength affect the PCNA nuclear protein
antigenicity involved in DNA synthesis. If enzyme digestion methods are used, the opti-
mal concentration of the enzyme (e.g., protease) must be applied. Unlike sodium citrate
buffer, enzyme solutions cannot be used at a range of concentrations. Note that the
concentration of the diluent used for primary monoclonal antibodies does affect the speci-
ficity and intensity of immunostaining (see page 82). It should be noted that the concen-
tration of the antibody also affects the specificity and intensity of immunostaining
(see page 80).
Factors Affecting Antigen Retrieval 75
Several antigen retrieval fluids are in use, all having been reported to efficiently medi-
ate antigen retrieval. A fluid applicable to all antigens is not available. The main reason is
the enormous variety of chemical structure of not only antigens but also epitopes of any
one antigen. In fact, the chemical nature of the epitope plays a key role in the effectiveness
of an antigen retrieval fluid. In other words, the tissue, cell, and antigen types determine
the retrieval fluid. This is substantiated by the fact that generally each type of epitope
determines the fluid type conducive to its maximal retrieval. A few examples follow.
Two antigen retrieval fluids, sodium citrate buffer (0.1 M, pH 6.0) and glycine-HCl
buffer (0.05 M, pH 3.5) containing 0.01% EDTA, were compared for their effectiveness in
unmasking a wide variety of antigens (Imam et al., 1995). Glycine-HCl buffer-EDTA
yielded stronger immunostaining of p53, androgen, estrogen, progesterone, and Ki-67,
whereas sodium citrate buffer produced superior immunostaining of vimentin and leuko-
cyte antigens. PCNA was unmasked equally well with either of the two antigen retrieval
buffers, while the two buffers were ineffective in retrieving antigens such as prostatic acid
phosphatase and pan-keratin. According to another study, compared with sodium citrate
buffer, Tris-HCl buffer (pH 9.5) containing 5% urea yielded more intense staining of
Ki-67 in mouse lung tumors (Ito et al., 1998). However, low background staining is likely
when using the latter antigen retrieval buffer. Certain other types of antigens require a
combination of antigen retrieval fluids or systems for their optimal retrieval. Methods
using such combinations are given in this volume. Comparative effects of antigen retrieval
systems on antigens are summarized in Chapter 6.
In addition to the chemical structure of antigens, a number of other factors, including
pH, heating temperature, molarity, and the chemical composition of the retrieval fluid, are
considered for selecting the optimal retrieval fluid. Optimal immunostaining of a given anti-
gen requires an antigen retrieval fluid of a specific pH. Note that optimal antigen retrieval
requires an optimal fixation procedure. Although the exact mode of action of antigen retrieval
fluids is not known, their salts may modify the hydrophobicity of polypeptide chains, affect-
ing the conformation of protein molecules. The major effect of the salts is to mediate high
temperature effects. However, this mechanism does not explain the mode of action of non-
buffer fluids (e.g., water) used for antigen retrieval. Excellent p53 immunostaining in breast
tumors has been achieved by heating the sections in water for 15 min at 50°C (Katoh and
Breier, 1994). Heating is carried out in a microwave oven or in a water bath.
Antigen retrieval can occur under both acidic and alkaline conditions, depending on
the type of antigen involved. The mechanisms involved in the antigen retrieval at different
pH values are not known. Three commonly used antigen retrieval fluids are 0.01 M sodium
citrate buffer (pH 6.0), 0.01 M Tris-HCl buffer (pH 1.0) or 0.1 M Tris-HCl (pH 10.0), and
0.05 M glycine-HCl buffer (pH 3.6). The latter can be used with or without 0.01% EDTA
depending upon the antigen type. Taylor et al. (1996b) recommend 0.1 M Tris-HCl buffer
(pH 9.5) containing 5% urea. These fluids provide strongly alkaline or acid environments
and are effective for antigen retrieval in tissues which have been either mildly fixed or
overfixed with formalin. These recommendations are based on the successful immunos-
taining of a wide variety of antigen-antibody complexes. For most clinical applications,
0.01 M sodium citrate buffer (pH 6.0) is recommended.
76 Chapter 4
Only if sodium citrate buffer or Tris-HCl buffer fail to yield satisfactory retrieval
should fluids containing substances such as EDTA, EGTA, enzymes, metal salts, periodic
acid, or urea be tried. Consider the immunostaining of parvalbumin, calbindin, and MAP,
which has been found to be best accomplished using 4% aluminum chloride (Evers and
Uylings, 1994b). However, in this study the antigen retrieval effect of the metal solution
was compared only with that of distilled water and zinc sulfate; sodium citrate buffer was
not tested. Since free-floating vibratome sections of the human brain tissue underwent
severe wrinkling during microwave heating, thick tissue slices (0.5 cm) were placed in 4%
aluminum chloride solution and heated in a microwave oven for 10 min, followed by stan-
dard immunocytochemical staining of semithin sections Another advantage of
pretreatment is that the brain tissue hardens, facilitating easier sectioning.
Fluids other than standard sodium citrate and Tris-HCl buffers are also preferred in
some other cases. An example is the retrieval of Bcl-2 antigen (oncoprotein), which is best
achieved by hydrated autoclaving of sections placed in deionized water (Umemura et al.,
1995). Immunostaining of neurofilament proteins, proliferating cell nucleus antigen
(PCNA), retinal S-antigen, and glial fibrillary acidic protein (GFAP) has been obtained by
using distilled water as the antigen retrieval fluid in a microwave oven (Yachnis and
Trojanowski, 1994). However, heating in water in a microwave oven is not generally rec-
ommended. Neurofilament proteins in archival tissues have been immunostained after
employing a saturated solution of lead thiocyanate (Yachnis and Trojanowski, 1994). Zinc
sulfate has been used for retrieving vimentin and prostate-specific antigen (Wieczorek
et al., 1997). Cesium chloride (5.7 M) has also been employed for antigen retrieval.
However, such metal salt solutions are not recommended because they are toxic. Target
unmasking fluid (TUF) was developed by van den Berg et al. (1993) for routine immuno-
histochemistry and is commercially available (Signet Lab, Delham, MA, or Kreatech
Biotechnology, Amsterdam).
Periodic acid (0.5%) has also been used as an antigen retrieval fluid (Xue et al., 1998).
Another type of antigen retrieval fluid is EDTA of pH 8.0 (Morgan et al., 1994; Pileri et al.,
1997), which has been stated to be effective irrespective of the location of the target mole-
cule (intranuclear, intracytoplasmic, or membrane-bound). Comparative studies by Ehara
et al. (1996) also indicate that EDTA (0.15 M, pH 6.0) yields stronger immunostaining of
steroid hormone receptors than that obtained with sodium citrate buffer (0.01 M, pH 6.0).
However, the preservation of cell morphology is superior when citrate buffer is used. Urea
(3 M), formic acid, and guanidine solutions have also been employed for antigen retrieval.
When urea is used in an autoclave or a pressure cooker, it has the disadvantage of yielding
false-negative results or background staining (Shi et al., 1996b). In another study, a satu-
rated solution of dimedone was applied for antigen retrieval (Shi et al., 1996b). Recently, it
was reported that the addition of calcium chloride to the antigen retrieval fluid of a low pH
improved the preservation of tissue morphology (Morgan et al., 1997a,b).
Boric acid (0.2 M, pH 7.0) in conjunction with low-temperature, heat-mediated anti-
gen retrieval technique has been successfully used as the antigen retrieval fluid for estro-
gen receptors on freshly cut sections of breast tissue (Peston and Shousha, 1998). Boric
acid is also very effective in antigen retrieval on the archival hematoxylin-eosin-stained
lymphoid sections on coated or uncoated slides, using conventional heat-mediated antigen
retrieval method (Biddolph and Jones, 1999). Lymphoid sections tend to dislodge from the
coated or uncoated slides in the presence of sodium citrate buffer during antigen retrieval.
Factors Affecting Antigen Retrieval 77
The above discussion indicates that although 0.01 M sodium citrate buffer (pH 6.0) is
commonly used, it is not a universally ideal antigen retrieval fluid for all types of tissues
and antigens. If published information is not available with regard to the best antigen
retrieval fluid for the antigen under study, the ideal retrieval fluid for each type of epitope
must be determined by trial and error.
The following four antigen retrieval fluids are commercially available (BioGenex,
San Ramon, CA). The approximate pH indicated below is valid at the time of manufac-
ture; the pH of the fluid may change during storage.
1. Antigen Retrieval Citra Microwave Solution used at pH 6.0.
2. Antigen Retrieval Citra Plus Microwave Solution used at pH 6.1.
3. Antigen Retrieval Glyca Microwave Solution used at pH 3.5.
4. Antigen Retrieval AR-10 Microwave Solution used at pH 10.5.
Other commercial sources for antigen retrieval fluids are:
1. Dako TRS, Dako Corporation, 6392 Via Real, Carpinteria, CA 93013 HIER
buffer, Ventana Medical Systems, Tucson, AZ
2. Target Unmasking Fluid (TUF*), Monosan (Sanbio), Fronstraat 2A, Postbus 540,
AM Uden, NL-5402, The Netherlands; Serotec Ltd., 22 Bankside, Station
Approach, Kindlington, Oxford, Oxon OX5 1JE, U.K.
When other antigen retrieval methods fail, antigen retrieval can be accomplished in
90% glycerin solution using a hot plate with a magnetic stir rod. This approach is thought
to improve preservation of tissue morphology as well as efficient retrieval of some anti-
gens. Glycerin has the advantage of having a very high boiling point (290°C) and being
nontoxic, stable, and reusable. The stir bar maintains a constant and uniform temperature
throughout the antigen retrieval fluid, prevents hot or cold spots, and thus facilitates reli-
able and consistent results. This method can also be used in a conventional hot air oven.
Many slides in metal slide racks can be processed simultaneously in this oven. Glycerin
solution can also be used for antigen retrieval in Coplin jars in a microwave oven; an empty
space must be kept between the slides.
The glycerin method has been used for retrieving a number of antigen types, includ-
ing estrogen receptor (Beebe, 1999). It is especially useful for very small, fragile biopsies
such as prostate needle biopsies and bowel biopsies. Pure glycerin fails to bring about anti-
gen retrieval, which means that water and heat are required to cleave the formaldehyde
molecule from the proteins, break down the methylene bridges, or rehydrate the proteins.
The exact role of glycerin in the antigen retrieval mechanism is not known. Further testing
of the usefulness of this procedure is awaited.
*Contents: chromium potassium sulfate dodecahydrate, sodium dodecyl sulfate, dextran sulfate, formamide,
phosphate salt, magnesium sulfate, pepsin, polyethylene glycol, and Triton. It has a low toxicity and is irritat-
ing to the eyes and skin. It is a colorless, nonviscous liquid.
78 Chapter 4
Procedure
The antigen retrieval fluid consists of a mixture of 100 ml of 90% glycerin and 10 ml
of Dako’s citrate buffer. The slide rack is placed in a bowl of an appropriate size and shape,
so that the stir bar rotates freely under the slide rack. A sufficient volume of antigen
retrieval fluid is transferred to the bowl ~ 1 cm above the level of the slides. The hot plate
is turned on and adjusted until the temperature of the fluid reaches 100–120°C; the dura-
tion of heating varies between 5 and 20 min. For estrogen and progesterone retrieval the
duration of heating (120°C) is ~7 min with a 15-min cool-down period before the slides
are transferred to distilled water for further processing.
Alternatively, such high temperatures in the bowl can be achieved by placing it in a
microwave oven, then removing and placing it on the hot plate. This is followed by adding
the stir bar and the slide rack to the bowl. It takes 1 min for 100 ml of the glycerin solu-
tion to attain a temperature of 125°C in a 600 W microwave oven on high. If more than
100 ml of solution is to be heated to the same temperature, for every additional 100 ml an
additional 1 min is required.
In addition to heating, retrieval fluid pH plays a key role in achieving optimal antigen
retrieval. It is thought that the pH is more important than the composition of the retrieval
fluid. This is supported by the demonstration that optimal staining of antigen SMI-32 was
achieved at pH 2.5 and 2-hr microwave heating at 90°C, whereas staining of antigen MAP-
2 was best obtained at pH 4.5 and 10-min full-power heating; in both cases 0.05 M citrate
buffer was used (Evers and Uylings, 1994a). Therefore, in optimizing the antigen retrieval
protocol, pH is a priority.
Note that there is no universally optimal pH for a retrieval fluid. The retrieval of each
type of antigen requires a specific fluid pH, although exceptions occur with antigens that
can be retrieved at a wide range of pH levels. For example, AEI and NSE (cytoplasmic
antigens), PCNA (nuclear antigen), and L26 and EMA (cell surface antigens), can be
retrieved at pH levels of 1.0–10.0 (Shi et al., 1995a). On the other hand, some antigens
(e.g., estrogen receptor) display a marked decrease in immunostaining at pH 3–6, while
still other antigens, such as cytoplasmic HMB45, show weak or negative staining at
pH 1-2 but excellent results in the high pH range (Shi et al., 1995a).
Some antigens are retrieved only at a low pH. These types are exemplified by throm-
bospondin and SMI-32 (neurofilament protein), which require pH levels of 1–2 and 2.5,
respectively (Grossfeld et al., 1996; Evers and Uylings, 1994a). Note, however, that pH
levels lower than 3.0 can severely damage tissue morphology, especially with intense heat-
ing. Low pH levels can also alter the localization of some cytoplasmic antigens, resulting
in false-positive staining of the nucleus. For example, an antibody (UCHL1) to T cell anti-
gen is effective at pH 6.0 but results in the staining of every nucleus at pH 2.0 (personal
communication, H. Y. Lan).
Generally Tris-HCl buffer produces better results at higher pH levels (e.g., pH 10.0)
than do some other buffers. On the other hand, sodium citrate buffer increases the intensity
and extent of immunostaining of a wide variety of tissue antigens at pH 6.0. EDTA-NaOH
(1 mM) at pH 8.0 also yields satisfying results. Although relatively high pH solutions, such
Factors Affecting Antigen Retrieval 79
as sodium citrate or Tris-HCl, are suitable for most antigens, low pH solutions are pre-
ferred for nuclear antigens (Taylor et al., 1996a). However, solutions of low pH generally
tend to cause weak focal background staining and damage to some epitopes. A test battery
based on three pH values (low, middle, and high) should be carried out to establish an
optimal protocol, including pH, for immunostaining previously unexamined antigens
(page 104).
The ionic strength of the fluid in which tissues are suspended during fixation with
formaldehyde, unlike retrieval fluid concentration, does influence antibody access to intracel-
lular antigens such as proliferation cell nuclear antigen (PCNA), nuclear protein (Ki-67)
detected by MIB-1 antibody, and nuclear antigen p120. Ionic bonds are known to be respon-
sible for a major portion of protein-protein interactions, and their breakage causes dissocia-
tion of the interacting proteins, resulting in increased detectability of the antigen. Such
breakage occurs with increased salt (NaCl) concentrations. It has been shown that the
immunofluorescence of antigens such as PCNA is increased when the cells are fixed in the
presence of increased salt concentrations (Bruno et al., 1992). The increase is greater for cells
in the phase of the cell cycle than for cells in S or phase. High salt concentrations loosen
the proteins, which are then stabilized with formaldehyde. In other words, increased ionic
strength weakens intra- and intermolecular ionic interactions during the process of crosslink-
ing with formaldehyde. Using the optimal ionic strength of the solution, which must be cus-
tomized for a given antigen, will facilitate the accessibility of the antibody to the epitope.
ANTIBODY PENETRATION
formaldehyde over glutaraldehyde, since the latter forms strong protein crosslinkages. It is
also well established that tissue specimens that have been fixed for a long time (weeks or
months) require more vigorous treatments of sections for antibodies to penetrate and have
access to the antigens. It is interesting to note that an antibody against a specific epitope
less sensitive to aldehyde fixation can be obtained by immunizing the mice with an anti-
gen in which the aldehyde-sensitive epitope has been blocked or altered.
To increase the penetration of antibodies into thin resin sections of fixed tissues, simul-
taneous heating of sections and antibodies has been attempted. This treatment is thought to
increase the labeling of certain antigens, whereas that of some other antigens remains unaf-
fected. It has been demonstrated that such a treatment enhanced labeling density by the anti-
amylase antibodies, whereas labeling with anti-DAMP antibodies remained unchanged
(Chicoine and Webster, 1998). Further developments of this protocol are awaited.
ANTIBODY DILUTION
Not only the type (e.g., the cell clone) and the source of availability of an antibody
but also its dilution are important in fully utilizing the effectiveness of an antibody as a
powerful tool to detect antigens. The optimal antibody concentration for antigen varies,
depending on whether the tissue used is aldehyde-fixed or frozen; generally higher anti-
body concentrations are required for sections of aldehyde-fixed tissues (Fig. 4.1).
Also, different forms of an antigen require different concentrations of the antibody for
their maximal detection. This is exemplified by the PC-10 primary antibody, which identi-
fies PCNA antigen at a dilution of 1:1000 in epithelial cells in normal colon tissue, whereas
a dilution of 1:400 is required to localize these proliferating cells in adenomatous polyps
(Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detected
in various tissue types at the MIB-1 dilution of 1:50, using the microwave heating antigen
retrieval method. However, in a few studies MIB-1 dilutions of 1:20 to 1:100 have been used.
There are many pitfalls, including false-positive and false-negative staining, to using
antibody concentrations of higher or lower than optimal concentrations. Labeling speci-
ficity partly depends on the antibody dilution, while background staining critically
depends on its dilution. Also, the esthetic appeal of the images produced by immunohis-
tochemistry diminishes with suboptimal dilutions of antibodies. Therefore, to avoid
unwanted staining, pay careful attention to the optimal working dilution of an antibody,
especially of a polyclonal antibody, and to washing procedures. Also note that antigen
retrieval treatments allow the use of increased dilutions of the antibodies. For example, for
sections of formalin-fixed and paraffin-embedded tissues, the optimal dilution of PC-10
antibody without heat pretreatment is 1:10 compared with 1:600 after microwave heating
(Haerslev and Jacobsen, 1994).
A fairly high concentration of the primary antibody is necessary to follow saturation
kinetics. However, the majority of these antibodies exhibits a bell-shaped concentration-
binding curve, with the binding increasing up to a specific antibody concentration and then
decreasing. Such a bell-shaped curve is due to unstable binding of the antibody to the anti-
gen under very high antibody concentrations. Effects of a high antibody concentration can
be examined with the method of Raivich et al. (1993). In practice, however, one rarely
Factors Affecting Antigen Retrieval 81
Both the pH and osmolarity of the diluent buffer (especially the type of solute) affect
the affinity of monoclonal antibodies for the antigen. It is known that electrolytes exert a
profound effect, not only on the structural relations of protein molecules, but also on the
reactivity of proteins (Hayat, 2000a). The reactivity of both monoclonal and polyclonal
antibodies with antigens is affected by the type of antibody diluent used. This is true
whether or not a heat-induced antigen retrieval is used. Optimal pH increases the sensitiv-
ity (staining intensity) as well as the specificity of immunostaining. Acceptable shelf-life
of antibodies can also be achieved at optimal pH and dilution in the presence of stabiliz-
ing protein (Boenisch, 1999). Unfavorable pH diminishes immunoreactivity because it
reduces antibody affinity for the antigen. The role of pH in the interaction between the
antibody and the antigen in immunohistological processing is explained below.
Antibodies are attracted to the epitopes of most glycoproteins and polypeptides ini-
tially through electrostatic charges and subsequently through van der Waals and
hydrophobic interactions (Boenisch, 1999). In immune reactions, the isoelectric point (pI)
of both antigens and antibodies is therefore of importance. The pI of polyclonal IgGs
ranges from 6.0–9.5. Monoclonal antibodies of at least this class possess an equally
wide range of individual pI values. If the pH of the diluent and/or solute is used in the same
range, the result will be changes in both electrostatic charge and conformation of at least
some monoclonal antibodies and possibly of some reactive epitopes. Antibody configura-
tion controls spatial complementarity. All these changes contribute to variable attraction
between the antibody and the antigen.
As stated above, the pH of the diluent affects the electrostatic charge of monoclonal
antibodies and thus the interactions between the antibody and the reactive epitope.
Consequently, the optimal operational pH of the monoclonal antibody is determined by the
electrostatic charge of the paratope and that of the epitope. Most effective initial attraction
between the paratope and the epitope occurs at the pI intermediate to the antigen and that
of the antibody. For most antibodies, but not all, this pH is mildly acidic (6.0). An increas-
ingly higher pH of the diluent will decrease the net positive charge of most monoclonal anti-
bodies, resulting in their reduced attraction to negatively charged target epitopes. Higher pH
values will also increase the hydrophobicity of antibodies, lessening the interaction between
the antigen and the antibody because of the decreased penetration by the latter.
It is suggested that new monoclonal antibodies be tested in several dilutions higher
than those recommended by the vendor, using 0.05 M Tris buffer (pH 6.0 and 8.6)
(Boenisch, 1999). The highest dilution and the pH at which maximal staining occurs
should be determined for routine use of the new antibody. This approach frequently allows
for the use of antibody dilutions much higher than those recommended by the supplier.
Factors Affecting Antigen Retrieval 83
Note that the use of higher concentrations of monoclonal antibodies does not improve
weak staining in immunohistochemistry because of paucity of or masked antigens.
Although PBS is commonly used as the antibody diluent, this solution has certain dis-
advantages. Sodium ions in PBS tend to shield negatively charged epitopes, thereby dimin-
ishing the attraction of positively charged reactive sites on the antibody, especially at an
alkaline pH. Phosphate ions, on the other hand, promote hydrophobicity. Therefore, the
use of PBS and phosphate buffer for diluting the antibodies is not desirable. Accumulated
evidence indicates that the most suitable diluent for both monoclonal and polyclonal anti-
bodies is 0.05–0.1 M Tris buffer (pH 6.0) (Boenisch, 2001). The advantage of this pH
becomes clear when considering that most antigens and monoclonal antibodies possess
opposite surface charges at pH 6.0. Thus, to achieve optimal immunoreactivity, the pH of
the environment should be intermediate between the pH of the antigen and that of the
monoclonal antibody. Note that any change in the composition and pH of the diluent
affects the performance of both the antibody and the antigen.
In addition to the pH, the osmolarity of the buffer used to dilute the primary antibody
tends to influence the immunoreactivity of monoclonal antibodies. The changes in the
molarity of Tris buffer used for diluting monoclonal antibodies are expected to result in
changes in the immunoreactivity of antibodies. It has been reported that the higher the con-
centration of cations (e.g., ) in the buffer or the higher the pH in their presence, the
less the immunoreactivity of the monoclonal antibodies (Boenisch, 1999). However, poly-
clonal antibodies may not show such an adverse effect.
analyzer (Becton Dickenson Cell Analysis Systems, Mountain View, CA). Antigen
retrieval in both of these studies was carried out using microwave heating. If the preserva-
tion of androgen receptor antigenicity is a problem in a formalin-fixed archived tissue, one
way to circumvent this problem is to use archived fresh-frozen tissues. On the basis of their
study, Dash et al. (1998) have suggested that the use of this antibody for retrospective stud-
ies does not correlate androgen receptor status with prognosis or therapeutic response. A
decrease in immunoreactivity of p53 antigen in stored paraffin sections is discussed on
page 85.
out by Wester et al. (2000) and van den Broek and van de Vijver (2000). These studies and
my personal experience are reviewed below.
Only a few studies report that prolonged storage of sections does not adversely affect
antigen detectability. For example, according to Williams et al. (1997), long-term
(6 months at room temperature) storage of sections of tonsil tissue had no effect on the
reactivity of the five antibodies tested. In contrast, a vast number of other studies demon-
strate decreased staining of stored sections, especially when they are stored at room tem-
perature. It has been demonstrated, for example, that antigens such as p53 and Ki-67 (lung
and breast carcinoma) show lower staining after storage for 3 years at room temperature
than sections stored for the same period of time at 4°C or –80°C (Grabau et al., 1998).
Nuclear estrogen receptor in breast carcinoma also shows higher reactivity when deparaf-
finized sections are stored for up to 4 weeks in 10% sucrose in PBS at 4°C than that shown
by sections stored at room temperature for the same duration (Bromley et al., 1994). This
increased staining could be the effect of cold temperature and PBS in which the sucrose is
dissolved. It is also known that dissolved salt solutions unmask epitopes in formalin-fixed
and paraffin-embedded tissues.
It has also been demonstrated that the staining of p53 in mammalian ductal carcinoma
decreased after slides were stored for 2 months at room temperature, but the antigen loss
86 Chapter 4
was significantly less when slides were stored at 4°C (Jacobs et al., 1996). A gradual loss
of staining of Ki-67 was reported in the colon tissue when the slides were stored for
9–21 days at room temperature before staining, although a delay of 5 days did not dimin-
ish the staining (Holt et al., 1997). Maintaining cut sections refrigerated and protected
from light failed to prevent such a loss of MIB-1 immunoreactivity with time. On the other
hand, when sections were stored for as long as 1 month at room temperature before stain-
ing of PCNA antigen in the same tissue, no adverse effect was observed on the immunore-
activity of PC10 antibody with this antigen.
According to Shin et al. (1997), p53 immunoreactivity was not decreased with stor-
age of slides for as long as 25–48 months at room temperature, provided staining intensity
is not the only objective of the study. The percentage of positivity of microwave-enhanced
immunoreactivity of p53 stored at room temperature and fresh paraffin sections was not
statistically significant. Nevertheless, the staining intensity of heated, stored sections was
stronger than that in nonheated, freshly cut sections. This study was carried out using
tissue blocks of head and neck squamous cell carcinomas stored for 4–15 years and lung
carcinomas stored for 14–25 years. Zinc sulfate (1%) was used as the antigen retrieval
fluid and was heated for 3 min in the microwave oven. Similarly, the immunostaining of
p53 antigen in sections of colorectal carcinoma stored for 6–14 months at room tempera-
ture was excellent after microwave heating (Kato et al., 1995). The aforementioned dis-
agreement is due to the study of p53 antigen in different tissues and/or differences in the
details of the methodologies used in different laboratories.
There are many reasons for the lack of consensus on this highly complex phenome-
non, and they are discussed below. Various studies mentioned above were conducted using
different parameters of antigen retrieval methods, including antigen retrieval fluids, pH,
heat source, temperature, and duration of treatments for detecting different antigens. The
type of fixation and duration of fixation also varied in these studies. Other variants were
the type of epitope and antibody and source of antibodies used.
The degree of immunostaining of stored paraffin sections may differ, depending on
whether monoclonal or polyclonal antibodies are employed. Polyclonal antibodies have
affinity for several types of epitopes, resulting in positive staining, which may be nonspe-
cific. An antigen retrieval method unmasks more than one type of epitope on the same
section, whether stored or not, and such epitopes have access to the polyclonal antibody.
However, a recent study indicates that polyclonal antisera show only slightly better
staining than that obtained with monoclonal antibodies (van den Broek and van de Vijver,
2000). It is also possible that loss of immunostaining in stored sections is epitope related
instead of related to the antigen as a whole (Henson, 1996).
In addition, tissue heterogeneity at different levels was not considered. The quantity
and quality of antigen reactivity varies from one tissue block to another. In some of these
studies the automated immunostainer was used, whereas in others manual staining was
carried out. The automated stainers reduce human procedural errors, and their controlled
environment increases the speed and timeliness of results in high-volume laboratories,
although their high cost might be prohibitive for small laboratories. Another factor that may
affect results is that a collection of stored sections may be heterogenous regarding dura-
tion of storage because of the successive addition of new sections. Subjective evaluation
or inadequate quantification of the extent and intensity of staining was the common
Factors Affecting Antigen Retrieval 87
denominator in most of these studies. These are the main reasons for the contradictory
results reviewed above.
Attempts have been made to protect sections from exposure to oxygen during storage
by coating them with paraffin (Jacobs et al., 1996; Rittman, 2000). This is accomplished
by heating the slide to ~60°C and placing a few drops of molten paraffin on it; a second
heated slide is gently drawn across the surface of the first slide to form a thin protective
paraffin layer on the sections. In our experience, coating the surface of paraffin sections
mounted onto slides with paraffin does not significantly reduce antigen loss after their stor-
age for several weeks or months at room temperature or at 4°C. Xylene-based spray glue
has also been used for protecting the stored sections but without success (Wester et al.,
2000).
Different antigens are affected differently by the storage of sections of even the same
formalin-fixed, paraffin-embedded tissue. In other words, the degree of the antigenicity
loss due to storage of the unstained slides differs depending on the type and location of the
antigen. For example, nuclear steroid receptors tend to be comparatively more sensitive to
storage (aging). Comparative studies, using a panel of eight antibodies against Ki-67,
prostatic-specific antigen, androgen receptor, epidermal growth factor receptor, and pro-
static acid phosphatase, demonstrated that nuclear androgen receptor showed a higher
decrease of antigenicity in stored, unstained sections compared with that exhibited by
other antigens (Olapade-Olaopa et al., 2001).
The loss of antigens due to storage of paraffin sections is not a serious problem in
many clinical immunohistochemistry laboratories that perform immunostaining within
hours or days after paraffin sections have been cut. However, antigen loss may become a
problem when slides are stored for months at room temperature as positive controls. Such
storage is encountered in some research laboratories where unstained paraffin sections are
archived for future use. In any case it is recommended that sections be stained rapidly after
they have been cut from the formalin-fixed, paraffin-embedded tissues. It is likely that pro-
longed storage of sections at room temperature strengthens protein crosslinks, which
become less reversible, resulting in diminished antigen retrieval. If immunostaining needs
to be postponed, tissue specimens should be stored in paraffin blocks rather than as paraf-
fin sections because antigenicity is better preserved in the former state. The results of a
comprehensive study on the effects of fixation, temperature and duration of section
storage, and antigen retrieval on the immunostaining of p53 antigen are shown in
Fig. 4.3 (Plate 2). Color-based image analysis was used to quantify the extent and inten-
sity of staining.
In conclusion, the storage of paraffin sections decreases, to a varying degree,
immunoreactivity for most, but not all, antigens. The maximal decrease in immunoreac-
tivity, at least of p53 and Ki-67 antigens, occurs during the first 2 weeks of storage (Wester
et al., 2000). The decrease in immunoreactivity is generally inversely related to an increase
in storage temperature. Both the extent and intensity of staining tend to be negatively influ-
enced by storing the sections. The decreased immunoreactivity as a result of section stor-
age can be compensated for in most cases by using optimal antigen retrieval procedure.
Although longer durations of fixation are accompanied by increased masking of antigens
during section storage, this relationship is neither universal nor linear. If paraffin sections
must be stored, they should be stored at –20°C, irrespective of the duration of storage.
88 Chapter 4
Factors Affecting Antigen Retrieval 89
SIGNAL AMPLIFICATION
Most antigens are not destroyed during fixation with formaldehyde, but are reversibly
masked. Methods to unmask them are presented in this volume. However, some antigens
are difficult to visualize adequately with routine immunohistological techniques and there-
fore require signal amplification with an acceptable signal-to-noise ratio. An amplification
of immunostaining intensity is especially useful when monoclonal antibodies are used
because they bind only a single epitope. Small amounts of antigens in tissue sections can
be detected specifically by using signal amplification. Techniques used for increasing the
sensitivity or signal amplification are summarized below.
A number of strategies have been employed for improving immunohistochemical sig-
nals. The immunofluorescence antibody method was developed for specific identification of
cells based on their antigen makeup (Coons et al., 1941). Its use is limited because of the need
for fresh-frozen sections and inadequate preservation of cell morphology. Also, the fixed ratio
of fluorescein to the antibody does not allow amplification of the signal. The peroxidase-
labeled antibody method is more compatible with the basic substrates of surgical pathology
specimens fixed with formalin and embedded in paraffin (Nakane, 1968). This immunoper-
oxidase protocol can be amplified by increasing the duration of development.
The original immunoenzyme bridge method using enzyme-specific antibody (Mason
et al., 1969) has been superseded by an improved technique using a soluble peroxidase
antiperoxidase complex (PAP) (Sternberger et al., 1970). These complexes are formed
from three peroxidase molecules and two antiperoxidase antibodies and are used as a third
layer in the staining method. They are bound to the unconjugated primary antibody (e.g.,
rabbit antihuman IgG) by a second layer of bridging antibody (e.g., swine antirabbit
immunoglobulin), which is applied in excess so that one of its two identical binding sites
binds to the primary antibody and the other to the (rabbit) PAP complex. The PAP method
is more sensitive than indirect methods using fluorescein or peroxidase-conjugated antis-
era. Alkaline phosphatase antibodies raised in the mouse can, by the same principle, be
used to form alkaline phosphatase anti-alkaline phosphatase (APAAP) complexes. These
have uses and advantages similar to those of the PAP complexes.
The avidin-biotin methods rely on the marked affinity of the glycoprotein avidin for
biotin. Avidin is composed of four subunits which form a tertiary structure possessing four
biotin-binding hydrophobic pockets. The oligosaccharide residues in avidin give it some
affinity for the tissue components, especially some lectinlike proteins, and result in non-
specific binding. A similar molecule, streptavidin, has some advantages over avidin, as the
former lacks oligosaccharide residues and possesses a neutral isoelectric point.
The low-molecular-weight vitamin biotin is easily conjugated to antibodies and
enzyme markers. Up to 150 biotin molecules can be attached to one antibody molecule,
and the strong affinity of the biotin for the glycoprotein avidin allows its use as complex-
ing secondary reagents. Biotin labeling of the primary (direct) or secondary (indirect) anti-
body can be used in the avidin-biotin methods. In the labeled avidin method the tracer is
attached directly to the avidin molecule. In the avidin-biotin bridge method a biotinylated
enzyme such as peroxidase is allowed to bind after attachment of avidin to the biotin-
labeled antibody.
In the avidin-biotin (ABC) method a complex of avidin and biotinylated tracer
containing the free avidin binding sites is applied to the biotinylated antibody. As a high
90 Chapter 4
limited to two or three. It should be noted that because the tyramide deposition reaction is
rapid, small differences in amplification duration may lead to variations in the final signal
intensities. Furthermore, because this reaction amplifies both specific and nonspecific
immunohistochemical signals, it is essential that appropriate positive and negative controls
be used to achieve correct interpretation of staining. Also, because TSA-ABC tends to
enhance the background noise along with the signal, the procedure must be optimized to
ensure low nonspecific binding. All tyramide conjugates yield approximately the same
92 Chapter 4
results, indicating that signal amplification is independent of the tyramide conjugate used
(Speel et al., 1998). Numerous biotin conjugated tyramides can be detected with avidin-
conjugate (Totos et al., 1997). If biotin reaction fails, the primary reason is the age of the
biotin solution. Because the shelf-life of newly synthesized biotin is not known, one should
at least be aware of the expiration date of the reagent.
Different authors and commercial suppliers have assigned different names to signal
amplification using tyramine. For example, tyramine signal amplification (TSA) system
and the catalyzed signal amplification (CSA) system are commercially available from
DuPont NEN Life Science Products, Boston, MA, and DAKO Corporation, Carpinteria,
CA, respectively. In addition, the terms CARD (catalyzed reporter deposition) (Bobrow
et al., 1989), TA (tyramide amplification) (Shindler and Roth, 1996), and ImmunoMax
(Merz et al., 1995) have been used for the tyramine amplification technique. The use of
different names for almost identical procedures has resulted in confusion. To standardize
the terminology, the neutral abbreviation, tyramide amplification technique (TAT) should
be accepted (Von Wasielewski et al., 1997).
One of the variations of the tyramide amplification technique is termed ImmunoMax
(Merz et al., 1995). In this approach the biotinylated tyramine enhancement is combined
with an antigen retrieval method such as microwave heating, enzyme (proteinase K) diges-
tion, or exposure to a detergent (guanidine hydrochloride). This method is effective in
detecting some previously unreactive, inadequately reactive, or partly demasked antigens
in the formaldehyde-fixed and paraffin-embedded tissues. It has been claimed that this
technique allows as much as 10,000-fold dilution of the primary antibody and 100 to
1,000-fold increase in sensitivity compared to those used with the conventional ABC
method (Merz et al., 1995). However, the sensitivity increase in the range of 5- to 50-fold
is more feasible (Speel et al., 1999).
This problem can also be lessened by using stronger fluorochromes and chemiluminescent
substrates for use in ELISAs, immunofluorescence-based staining, and immunoblotting.
Detection of low concentrations of antigens can also be achieved by increasing the
signal without raising the level of nonspecific background staining. Signal amplification,
for example, can be achieved by successive steps of enzymatic reactions. Biotinyl tyramide
is commonly used to increase the signal of low abundance targets that are otherwise unde-
tectable by conventional techniques. However, tyramide-based amplification may increase
background noise because of multiple steps of signal amplification (discussed in this chap-
ter). Therefore, molecular tissue pathology requires techniques of greater sensitivity and
specificity. One of such techniques to refine the examination of cell components is rolling
circle amplification (RCA) discussed below (Lizardi et al., 1998).
Rolling circle amplification is essentially a surface-anchored DNA replication that
can be used to visualize single molecular recognition events. It is an isothermal nucleic
acid amplification protocol that differs in several aspects from the polymerase chain reac-
tion (PCR) and other nucleic acid amplification methods. The RCA can replicate circular-
ized oligonucleotide probes with either linear or geometric kinetics under isothermal
conditions. It has sufficient sensitivity to detect individual oligonucleotide hybridization
events and single antigen-antibody complexes (Schweitzer et al., 2000). The linear mode
of RCA can generate signal amplification during a brief enzymatic reaction.
Another advantage of linear RCA is that the product of amplification remains connected
to the target molecule. Signal amplification by RCA can be coupled to nucleic acid
hybridization and multicolor fluorescence imaging to detect single nucleotide changes in
DNA within a cytological context or in single DNA molecules (Zhong et al., 2001). This
protocol has been used for visualizing target DNA sequences as small as 50 nts long in
peripheral blood lymphocytes or in stretched DNA fibers (Zhong et al., 2001).
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Chapter 5
LACK OF IMMUNOSTAINING
The failure of antibodies to immunostain tissues or cells does not necessarily reflect the
absence of epitopes. Lack of, or reduced, immunostaining can be attributed to multiple fac-
tors. The most common factor is the inability of the antibody to reach and recognize the
epitope under the preparatory conditions used, including fixation, dehydration, embed-
ding, deparaffinization, rehydration, and incubation. The inaccessibility of the epitope to
the antibody may be due to the formation of large, compact protein complexes as a result
of crosslinking by formaldehyde. These complexes create a barrier to antibody penetration.
This aspect of immunostaining failure is elaborated upon later. It is also possible that the
antigen molecule is folded and thus hides, the epitope, especially from monoclonal anti-
bodies. Apparently, better immunostaining depends on improving antibody access to, and
recognition of, the epitope.
It is well established that many types of antigen molecules are altered by dehydration
solvents and other reagents. Lack of immunostaining may also be due to excessively
diluted antibody, to loss of antibody owing to degradation by bacteria or fungi, or to anti-
body aggregation due to repeated freezing and thawing. Finally, a monoclonal antibody
will not recognize an epitope in vivo if the former is raised against a denatured antigen.
This is also true for polyclonal antibodies when the recombinantly produced antigen
becomes denatured during isolation and purification (Binder et al., 1996).
Fixation with aldehydes plays a key role in the two above-mentioned events: antibody
access to and recognition of the epitope. Tissues and cell cultures are usually fixed with an
aldehyde prior to immunostaining. Fixation has the advantage of anchoring in situ anti-
gens, as aldehydes are powerful protein crosslinking agents. They crosslink proteins and
glycoproteins through reversible and irreversible alterations in the molecular conformation
of proteins, including antigens (epitopes). If the change in conformation is strongly
irreversible, the antibody will have difficulty recognizing the altered epitope, especially
aldehyde-sensitive antigens. This problem is especially acute when specimens are fixed
with glutaraldehyde. This effect of aldehydes on epitopes and their surrounding proteins is
called epitope masking. Briefly, epitope unmasking can be accomplished by weakening or
breaking down the protein crosslinking introduced during aldehyde fixation and allowing
the epitope to be exposed to the antibody, provided the latter has access to the former.
Thus, two simultaneous events (unmasking of epitope and access of antibody to epitope)
95
96 Chapter 5
BACKGROUND STAINING
animal with adjuvants. Although these antibodies are difficult to remove, their net effect
can be almost eliminated by using the antiserum at a sufficiently high dilution or by reduc-
ing the duration of incubation.
Nonspecific staining may also result from contaminating antibodies produced by the
host’s immune system as it reacts to isolated antigens used for immunization (Boenisch,
2001). Isolated antigens are rarely pure. If these antibodies are a problem, the antiserum
should be subjected to affinity absorption. Fortunately, such antibodies are present in a
very low concentration and may not cause troublesome background staining. Use of high-
titered antisera at sufficiently high dilutions would eliminate this problem. Natural and
contaminating antibodies do not cause any problem when using monoclonal antibodies.
Nonspecific staining can be caused by Fc receptor glycoproteins present on the cell
membrane. This problem is more relevant to frozen sections and smears than to tissues
fixed with formaldehyde. The problem can be avoided by using fragments instead
of whole IgG molecules (Boenisch, 2001). Complement-mediated binding may also cause
background staining in frozen sections when whole antisera is used; however, this prob-
lem is not very common.
Antigen diffusion can cause specific background staining. This problem arises when
the target antigen is displaced from its site of synthesis or storage. Delayed fixation and/or
incomplete fixation with formaldehyde tend to cause this problem. Optimal fixation with
this monaldehyde anchors the antigens at their site of synthesis. Mechanical injury to the
tissue or drying of the tissue prior to fixation may result in diffuse background staining.
Necrotic areas due to autolysis of the tissue tend to stain with almost all staining reagents.
Antigen retrieval with prolonged enzyme digestion often disrupts cell architecture, result-
ing in the displacement of target antigens from their site of greater density; the net effect
is increased background staining.
Background staining also results from the presence of endogenous peroxidase in the
formalin-fixed tissues. This artifact can be avoided by treating the tissue sections with 3%
hydrogen peroxide in water for 4–9 min at room temperature; methanolic hydrogen per-
oxide is not recommended. Blocking of the endogenous peroxidase activity is especially
desirable with cell preparations and frozen sections (Boenisch, 2001).
Endogenous biotin, distributed in a wide variety of tissues, may also cause back-
ground staining with biotin-based immunohistochemical techniques. This biotin is espe-
cially abundant in liver, whereas it is poor in the central nervous system and adipose tissue.
Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but is
also present in sections of paraffin-embedded tissues. This problem is largely eliminated
by using streptavidin-based methods or by sequential treatment of sections (prior to stain-
ing) with 0.01–0.1% avidin followed by 0.001–0.01% biotin for 10–20 min each. The
biotin problem is discussed in more detail later in this chapter.
Other causes of diffuse background staining include the presence of residual embed-
ding medium and bacterial or yeast contamination in the water bath. The presence of
undissolved chromogen granules on occasion may create the problem of nonspecific stain-
ing. Excessive counterstaining with reagents, such as hematoxylin and eosin, may com-
promise specific staining.
Finally, a few published reports indicate that antigen retrieval at extremely high tem-
peratures may result in nonspecific staining. Baas et al. (1996), for example, have reported
false-positive results at a very high antigen retrieval temperature using monoclonal
98 Chapter 5
antibody D07 against p53 antigen. They carried out antigen retrieval at 96°C for 30 min in
the Target Unmasking Fluid (TUF) containing 35% urea in a microwave oven. This com-
bined treatment is unusually excessive and is not used routinely.
Procedure
All the reagents are commercially available in kit form (polyMICA: Binding Site
Ltd., Birmingham, UK).
Microwaves consist of electric and magnetic fields, and they propagate in space.
Electric fields are primarily responsible for physical effects on the tissue. The energy dis-
tribution and thus the speed of absorbing energy and warming up vary topographically. In
Problems in Antigen Retrieval 103
other words, the spatial energy distribution in a microwave oven is unequal. A region in
the oven with a high intensity of electromagnetic fields is known as a hot spot or, more cor-
rectly, a hot region (~1 cm).
Hot areas are not located at fixed coordinates in the oven and are influenced by the
load placed in the oven. Loads of various sizes and shapes lead to different heating pat-
terns. By varying the position of the load within the oven during irradiation, reproducible
results can be obtained. The problem of hot areas can be solved by providing a microwave
transparent rotating platform for the load during irradiation and by placing an extra load to
serve as a heat sink; a jar containing 100 ml to 1 liter tap water or antigen retrieval solu-
tion suffices. To obtain reproducible results the same type of jar should be placed in the
same location in the oven. For detailed theoretical and practical considerations of hot
areas, the reader is referred to Kok et al. (1993).
the fixative, temperature and duration of fixation, size and composition of the tissue, and the
amount of free blood. Also, irrespective of the size of the tissue block, the fixation is vari-
able within the block. The same duration of fixation with formaldehyde may or may not
result in identical fixation, for each tissue block responds uniquely to the fixative.
Moreover, uniform sections are difficult to obtain, and their thickness is difficult to
determine. Many sections are wedge-shaped rather than planoparallel (Rittman, 1998). In
addition, the arc of vibration caused by the knife edge as it cleaves the section is sufficient,
for example, to glide over or under the surface of some nuclei or cut through the remain-
der. Consequently, an accurate measurement of the concentration of nuclear antigens is
difficult (Allison, 1999). The tendency is to cut the thinnest possible sections to obtain
superior resolution that provides distinct images. However, thin sections show excessive
compression as well as variation in thickness because compression is usually inversely
proportional to section thickness. Another obstacle is the small number of sections that are
usually examined in a diagnostic laboratory, limiting the production of reliable average or
quantitative data.
TEST BATTERY
controls. Appropriate positive and negative controls, as well as the study of fresh-frozen
tissue sections, are required to rule out any false-negative or false-positive staining.
Duplicate immunostaining will assess reproducibility.
Pathologists play a key role in the diagnosis of cancer, and their histopathological
assessments are accepted as the gold standard. Although not admitted, the process by
which a pathologist makes a diagnosis is inherently subjective. A number of factors,
including clinical features of the lesion, the clinical impression offered by the surgeon, and
the training and experience of the pathologist, play a part in determining the final “sign-
out” diagnosis on which the final treatment decisions depend. These decisions have far-
reaching consequences in the quality of health care. It is obvious that no other type of error
in the medical profession is more important, less understood, and less frequently admitted
than fallibility in histopathological diagnosis. Kaugars (1995) has aptly pointed out that the
sign-out is written on paper, not on stone tablets.
Can the intraobserver and interobserver variations in diagnosis be eliminated?
Unfortunately, the answer is no. However, avoidable fallibilities must be avoided. The inter-
observer variations can be significantly reduced by a joint session behind a microscope in
a process of “practical agreement” (Vet et al., 1995). Prior to such sessions, participating
pathologists reach consensus on the relevant pathological grading characteristics (theoreti-
cal agreement). A theoretical agreement increases the practical agreement between pathol-
ogists. Interobserver agreement is definitely improved when pathologists confront each
other’s observations and arguments. Even experienced pathologists will benefit by a joint
session behind the microscope.
Both low-power and high-power microscope observations are useful in markedly min-
imizing interobserver variation. The characteristics observed at low magnification include
atypia, location of immature cells, and stratification/polarization. At high magnification,
detailed morphological characteristics, such as location of immature cells and stratification/
polarization (differentiation), nucleus/cytoplasm ratio, hyperchromasia, polymorphous
nuclei (cell characteristics), and the location and appearance of mitotic activity, are scored
(Vet et al., 1995).
Another approach to avoiding interobserver and intraobserver variations and standard-
izing the diagnosis is the use of computer-assisted analyses. This technology is beginning
to be employed in some laboratories (see below).
QUANTITATION OF IMMUNOSTAINING
AUTOSTAINERS
kinetics and that produce results within 1 hr. Furthermore, the practice of manually staining
a large number of slides is tedious and time consuming. Typically, manual hematoxylin-eosin
staining (hematoxylin stains nuclei blue and eosin stains cytoplasm pink) is completed in
approximately 25 steps. Autostainers save a technologist’s time, which permits him or her to
carry out more technically demanding tasks. After the staining protocol has been standard-
ized, the stainer does not require the technologist’s attention during its operation.
There are many other advantages to using autostainers. They prevent risk of exposure
to certain hazardous reagents (xylene). Some stainers have built-in fume hoods or can be
used in a fume hood; in the former case, the need for a large overhead fume hood is
eliminated. Another advantage is consistency of the technique that eliminates intra- or
interpersonal variations in results. Also, a consistent temperature can be maintained for
temperature-sensitive procedures. The space requirement for some stainers (e.g., centrifu-
gal stainer) is smaller than that required for manual staining for some procedures. Efficient
stainers use reagents conservatively, thus reducing the amounts needed and the risk of
contamination.
There are a few limitations to equipping a laboratory with an autostainer. The high
cost of most stainers may be prohibitive for a small laboratory. Unavailability of space in
such a laboratory is also a possibility. Repairs of this machine are expensive and may take
a long time if the service technician lives in another state. Some stainers require the pur-
chase of prepackaged reagents that are also relatively expensive. Moreover, some technol-
ogists may object to being restricted to using these reagents. In addition, a staining defect
occurring during operation of the stainer is revealed only after staining is complete. On the
other hand, if a problem arises during manual staining, the process can be stopped and the
problem corrected. But above all, the use of a stainer limits the technologist’s understand-
ing of the actual staining process.
There are two types of autostainers: in the first type, the slides are immersed into
the reagent; in the second type, the reagent is applied to the slides. Stainers that immerse
the slide into the stain (bath stainers) can be either linear or batch design (Earle, 2000). The
linear type is based on a carrier mechanism that allows loading of the slides into the slide
holders (racks), one at a time, and their sequential immersion into the staining solution.
The slide holders are attached to the carrier, which moves at a uniform speed, and the
slides exit the stainer one at a time. This type of machine is long, processes
~360–720 slides per hour (12–14 min per slide), and requires water and a drain. It may
have a built-in fume hood and slide dryer. Batch stainers move slide holders, each con-
taining several slides, through baths of the staining solution. Programmable batch stainers
are now commercially available which use robotic arms to move the slide racks from one
position to the next. These stainers can be programmed to agitate the slides in the staining
bath. Simultaneous multiple staining can be accomplished in some machines based on this
principle. A combined linear-batch stainer is also available, which moves slide containers
through a series of stain containers. Each rack may hold a small or large number of slides,
and continuous staining is possible. The machine is long, processes 24–66 slides
per rack, and the time taken depends on the program. It may be compatible with the cover-
slipper, and some have built-in fume hoods. The machine may require running water and
a drain, and it may have waste collection.
There are three types of stainers that apply the reagents to the slides: capillary gap
stainers, centrifugal stainers, and flat-method stainers (Earle, 2000).
Problems in Antigen Retrieval 109
Capillary gap stainers are based on the principle that the staining solution is forced
between the slide and the area around it. Essentially, rotating gears move slides (face
downward) along a plane surface that has holes through which the stain is pumped at
appropriate intervals. The advancing slides press a switch as they pass each staining sta-
tion, thus activating a pump. The stain is discarded after the slide moves to the next sta-
tion, avoiding the contamination of the bulk containers. The machine pumps the stain from
the closed bulk containers to the plane surface via small tubing, minimizing reagent
evaporation.
The capillary gap system can also be used in stainers that use two slides face-to-face
to provide the capillary gap. Robotic arms move holders of paired slides to staining,
draining, and rinsing stations. Because this system uses very little staining solution, it is
recommended for immunostaining large numbers of slides. The machine is ~3 ft long,
may be compatible with a cover-slipper, and some may have a built-in fume hood. This
system requires prepackaged reagents and may require a drain.
Centrifugal Stainers
Centrifugal stainers spray the staining solution onto the slides as they rotate past the
spray nozzles in a spinning chamber. The prepackaged reagents are in closed containers
with pump tubing, which prevents evaporation and contamination of the chemicals. The
machine is smaller than 2×2 feet, stains 12 slides in 6–8 min, requires prepackaged
reagents, and may require a drain. It usually does not require a fume hood.
Flat-Method Stainers
Flat-method stainers drop staining solutions onto the slide as it lies flat within the
stainer. Some stainers employ robotic arms to apply solutions to the slides. This system is
in common use for immunohistochemical staining. The machine is long, stains
20–40 slides, depending on the system, in and slides require predeparaffinization.
The protocol may require prepackaged or manufacturer’s reagents and a waste container.
It is recommended for immunohistochemistry. For additional details about autostainers,
see Earle (2000).
The following automatic tissue processors and stainers are commercially available.
1. AP 280 Embedding Station, Carl Zeiss, Inc.
One Zeiss Drive, Thornwood, NY 10594
2. ATP1 Tissue Processor, Triangle Biomedical Sciences, Inc.
3014 Croasdaile, Durham, NC 27705–47770
3. Cytologix Stainer, Cytalogix Staining System
99 Erie Street, Cambridge, MA 02139
4. Lab Vision Auto Stainer, DAKO Corporation,
6392 Via Real, Carpinteria, CA 93013
110 Chapter 5
The volume-corrected mitotic (M/V) index can be used to test for differences between
borderline and malignant tumors. This index expresses mitotic activity as the number of
mitotic figures per square millimeter of neoplastic tissue in the microscope field. Usually
10 fields are counted at a magnification of 40, which corresponds to of neoplas-
tic tissue in the section. The M/V index has the advantage of not being influenced by the
size variation of the microscope field or cellularity of the neoplasm (Haapasalo et al.,
1989). Also, this method is easy, relatively rapid, reproducible, inexpensive, and available
to all pathologists (Miliaras, 1999). The morphometric formula of the M/V index renders
mitotic counts a more reproducible criterion because it avoids some of the limitations, such
as differences in microscope field size, of the conventional mitotic index. The M/V index
has been used for mitotic counts in many human neoplasms for both diagnostic and prog-
nostic purposes (Lipponen et al., 1990). Recently, Miliaras (1999) has used this index for
determining differences in p53 immunoreactivity and the proliferation rate between bor-
derline and malignant ovarian tumors.
The M/V index is evaluated as the number of mitoses per 10 hpf (high power field)
and is calculated according to the following formula proposed by Haapasalo et al. (1989).
Gleason grading is now the most widely used system for grading prostatic carcinoma.
This system is an effective tool for prognostication and as an aid in therapeutic decisions
for men with prostate cancer. The system is characterized by two major features: (1) it is
based solely on architectural pattern but cytological features are not evaluated (Gleason
and Mellinger, 1974) and (2) the overall grade is not based on the highest grade within the
tumor. The prognosis of prostate cancer is intermediate between the most predominant and
the second most predominant pattern of cancer (Fig. 5.3). Consequently, the grades of the
most prevalent and the second most prevalent pattern (~5% of the tumor) are added
together to obtain a Gleason score (Allsbrook et al., 1999). If the tumor shows only one
pattern, the pattern grade is doubled to obtain the Gleason score; for example, for all
pattern 3, the Gleason score is 6 (Fig. 5.4). The Gleason score is directly correlated with
mortality rates, is a predictor of time to recurrence after surgery, and of response to
therapy. Presently, the Gleason score, along with PSA and tumor stage, forms the database
upon which radical therapies are recommended (King, 2000).
The Gleason score alone suffers from interpretation bias and its accompanying grade
errors. Evidence is available indicating a lack of interobserver reproducibility of this score.
As expected, interobserver agreement is significantly better among pathologists who
learned Gleason grading at a professional meeting or course than among those who had
112 Chapter 5
not (Allsbrook et al., 2001a, b). The interpretation bias is significantly minimized through
a consensus pathological evaluation, while sampling effects are maximally reduced by
using an optimal number of biopsy cores. These two remedies, when applied in combina-
tion with the Gleason score, result in maximal grading accuracy. Another approach to
Problems in Antigen Retrieval 113
As stated earlier, lack of a standardized antigen retrieval method results in intra- and
interlaboratory variability in immunostaining results. The following method of microwave
Problems in Antigen Retrieval 115
oven calibration is a step toward obtaining reliable immunostaining (Tacha and Chen,
1994). This approach avoids repeated interruption of oven heating to replenish the antigen
retrieval fluid. The method essentially establishes the time to boiling point, after which the
setting is adjusted to maintain a simmering temperature. At such mild temperatures, the
separation of sections from the slide is less likely.
Place the jar containing 250 ml of antigen retrieval fluid and slides in the center of the
microwave oven, and set the oven on high power (800 W) for 2–3 min, until the fluid
begins to boil. Turn off the oven and record the exact time it took to achieve boil. Set the
oven on low power (~300 W) for 7–10 min, and adjust the setting so that the oven cycles
on and off every 20–30 sec and the fluid boils for ~5–10 sec/cycle. Also, note this setting.
The following formula can be used to determine the power setting:
S = 250/P × 10,
where S is the oven power setting and P is the output power of the oven. For example,
if the oven output power is 800 W, the power setting for antigen retrieval (S) will be
S= 250/800 × 10= 3.1. Therefore, set the oven on 3 and heat at 100°C for 7–10 min to
achieve antigen retrieval, depending on the antibody used.
Microwave ovens with temperature readouts are commercially available (Energy
Beam Science, Agawam, MA). Their power output is regulated by a temperature feedback
mechanism and timer, so that both temperature and time can be monitored. They can also
be used for fixation and accelerated immunostaining.
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Chapter 6
Antigen Retrieval
117
118 Chapter 6
for a prolonged time (e.g., 48 hr) or at higher temperatures (e.g., 80–100°C) for very short
durations, that is, in minutes. Specific peptide-bond cleavage by microwave heating in
weak acid solutions is a well-established method in protein chemistry (Wu et al., 1992).
The specific cleavage sites of peptide bonds are located at the carboxyl- and amino-terminal
ends of aspartyl residues along the peptide chain. Thus, heat can free epitopes from other
proteins and attached molecules.
That heat is not the only method to unmask epitopes is exemplified by enzyme diges-
tion or detergent treatment. The exact mechanism responsible for epitope retrieval with ultra-
sound is not clear, although intense heat is produced for an exceedingly short duration. It is
known, however, that ultrasound and/or heat decreases the amount of negative charges on the
cell surface (Joshi et al., 1983; Adler et al., 1988). Mechanical vibrations of molecules caused
by ultrasound and heat are thought to unfold the protein molecule and to expose the epitopes.
The mechanism underlying unmasking of epitopes with digestive enzymes is better
understood. Enzymes such as trypsin II, used in epitope retrieval, are powerful, tested
protein-digestive molecules. They are known to digest proteins and break down protein
crosslinkages introduced during formaldehyde fixation. As a result, the tight network sur-
rounding the epitopes is dismantled, allowing access of antibodies to the epitopes. If anti-
gen retrieval with protease digestion must be carried out, 100 mg of trypsin in 100 ml of
Tris-buffered saline (pH 7.8) can be used for 15–20 min at 37°C.
Antigen Retrieval 119
Like conventional heat, microwave heat breaks down protein crosslinks. The most
common explanation presented in the literature for unmasking epitopes with heat in the
microwave oven is hydrolysis of protein crosslinkages. However, the effects of microwave
heating on the tissue sections resulting in epitope retrievals are exceedingly complex. Heat
alone may not be enough to explain the effects of microwaves on epitope retrieval. This
view is supported by the observation that microorganisms are killed at lower microwave
temperatures or with shorter exposures than those required when conventional heat is used
(Chipley, 1980). Furthermore, because microwave heating also enhances immunostaining
of ethanol-fixed tissues, it is apparent that such heating unmasks epitopes by a mechanism
other than or in addition to breakage of protein crosslinks because this coagulative fixative
does not introduce crosslinks.
The above-mentioned evidence indicates that in addition to the direct thermal effect
of microwaving, microwave heating facilitates epitope retrieval by another simultaneous
mechanism. The microwave energy irradiated on the tissue sections in various liquid media
is lost or absorbed by the samples by two mechanisms: ionic conduction and dipole rota-
tion. Both effects occur simultaneously to account for the phenomenon of rapid heating
(Kingston and Jessie, 1988). It is thought that microwaves unfold protein molecules,
exposing the epitopes by subjecting the molecules, at least polar molecules such as water
and polar side chains of proteins, to rotational movement. As a result, these molecules
reach to a high energy level, unmasking the epitopes.
It is known that microwaves interact with dipolar molecules by (1) imparting kinetic
energy and raising temperature and (2) altering electric fields. Microwaves induce dielec-
tric fields, causing dipolar molecules to rapidly oscillate 180 degrees. In other words, these
molecules oscillate at the frequency of 2,450 MHz or at about 2.5 billion cycles per
second. Thus, microwave action is also due to rapid oscillation along the axes of asym-
metrical molecules such as water, proteins, and fatty acids, which behave as dipoles in an
attempt to reorient their positive and negative poles to keep up with the rapidly changing
electrical fields generated by microwaves (Salvatorelli et al., 1996). It has been shown that
the oscillating electric field causes cell poration (Chang, 1989). It is also known that
microwaves irreversibly alter the plasma membrane, with subsequent changes in ion trans-
port, breakdown of hydrogen bridges and secondary bridges, alterations in protein hydra-
tion, and release of bound water. All of these phenomena explain why microwaves exert a
different effect than that of conventional heat. It is apparent that not only the thermal but
also the nonthermal component of microwaves deserve consideration as effective epitope
retrieval factors.
Evidence indicates that microwaves affect the kinetics of conformation changes of
proteins such as (Bohr and Bohr, 2000). It is thought that even approxi-
mately a few GHz can excite protein molecules. Consequently, the kinetics of conforma-
tional changes of the protein molecule are enhanced, and this denaturing effect is
120 Chapter 6
nonthermal. In fact, microwave irradiation can cause folding or unfolding of protein mol-
ecules. However, additional evidence is needed to substantiate the role of the nonthermal
effect of microwave irradiation in antigen retrieval.
An understanding of the interaction between the antigen (epitope) and the antibody
visualized immunohistochemically can be attempted by considering at least the role of
formalin fixation, antigen retrieval fluid, and heat treatment. How each of these three factors
affects the molecular structure of the antigen or the epitope is the fundamental question. The
significance and relevancy of this question are apparent because an antibody recognizes
the corresponding epitope based on the molecular structure of the latter. The following dis-
cussion considers the possible role of calcium in masking antigens during fixation with
formaldehyde; it is based primarily on studies carried out by Morgan et al. (1994, 1997a,
b), Shi et al. (1997, 1999a), and Taylor et al. (1996a, b). The role of other factors in anti-
gen masking and retrieval is discussed elsewhere in this volume.
Endogenous calcium is an important factor in epitope masking. Biochemical studies
indicate that calcium binding induces a conformational modification of the protein mole-
cule, resulting in either a reduced antigen-antibody recognition effect (e.g., for throm-
bospondin) (Wilson, 1991) or the reverse effect (for protein C, a vitamin K–dependent
enzyme involved in blood coagulation) (Wakabayashi et al., 1986). It has been proposed
that removal of calcium by chelation significantly modifies the thrombospondin confor-
mation (Dixit et al., 1986). These changes may expose epitopes necessary for the binding
of certain monoclonal antibodies. This and other evidence indicates that calcium-induced
changes in the conformation of different proteins may result in negative or positive detec-
tion of immunogenicity. The ability of some monoclonal antibodies, but not all, to recog-
nize their corresponding epitopes is calcium-dependent under certain conditions. Different
antibodies respond differently to the calcium-induced modification of the same protein.
In relation to fixation with an aldehyde, possible mechanisms responsible for mask-
ing or unmasking epitopes as a result of tissue-bound calcium and calcium chelation,
respectively, are detailed below. One of the major effects of formaldehyde fixation is the
generation of a large number of hydroxymethyl groups through selective interactions with
various functional groups (e.g., active hydrogens on aromatic rings, primary and second-
ary amines, and hydroxyl and sulfhydryl groups) in proteins. The hydroxyl component of
the hydroxymethyl groups is thought to be reactive, depending on which of these functional
groups the formaldehyde is bound to. Such an active hydroxyl component could form a
coordinated bond with calcium ions (Fig. 6.2). Thus, proteins fixed with the aldehyde may
become complexed with calcium ions that are abundant in animal tissues, reaching levels
on the order of 2 mM in the cytoplasm of eukaryotic cells. These complexes mask epitopes
to a variable degree. Calcium complex formation with proteins in this state is likely to be
quite strong, involving four to eight coordinate bonds. Therefore, a considerable amount
of energy (heat) is required to release the calcium ions from this cagelike complex.
Based on this observation, calcium released from this complex requires high-
temperature heating in combination with a calcium chelating and/or precipitating agent
such as EDTA, EGTA, citrate buffer, or urea. Because these reagents are chelators of divalent
Antigen Retrieval 121
metal ions, epitope unmasking can be achieved by exposing the sections to such treat-
ments. Figure 6.3 shows the disruption of some coordinate bonds at a high temperature in
the presence of EDTA, which results in antigen retrieval. Also, an inorganic salt such as
sodium carbonate is expected to remove calcium by preferential precipitation. It is inter-
esting to note that microwave heating also causes changes in metal ion transport through
the plasma membrane.
122 Chapter 6
Further evidence supporting the role of calcium in antigen masking was provided by
Shi et al. (1999a). They exposed frozen tissue sections to 50 mM (pH 7.1) overnight
at 4°C and demonstrated a significant loss of immunostaining or altered staining pattern of
antigens such as thrombospondin and Ki-67 compared to controls not exposed to
Such a loss of staining can be partially recovered by incubating the sections in EDTA, sub-
stantiating the role of calcium in antigen masking. The antigen masking effect of calcium
has also been demonstrated by adding to antigen retrieval fluid such as EDTA (Kim
et al., 1999b). The disodium salt of EDTA (in common use) binds one divalent metal ion
only, and the addition of molar excess of calcium ions would eliminate the antigen retrieval
effect of EDTA.
The role of pH in calcium-related effects on antigen unmasking is controversial.
According to Morgan et al. (1997b), two different mechanisms are involved in antigen
retrieval at acidic and alkaline pH levels. Under acidic conditions (pH 1–3), instead of
chelation, high concentrations of hydrogen ions dissociate calcium complexes and/or
breakdown protein crosslinkages introduced by formaldehyde. On the other hand, in an
alkaline environment (pH 8.0), chelation of calcium is responsible for antigen retrieval and
can be carried out with a chelator. Dixit et al. (1986) had also proposed earlier that the
removal of calcium by chelation modifies the conformation of protein molecule as an
unrolling or unraveling of the large domains, resulting in the exposition of epitopes necessary
for the binding of certain monoclonal antibodies.
A somewhat different interpretation of the relationship of aldehyde fixation with the
masking of antigens with calcium-protein complexes is reported by Shi et al. (1999a).
According to this point of view, although calcium-induced modification of the protein
molecule does occur and can be demonstrated immunohistochemically, it is independent
of formalin-induced crosslinking. Addition of calcium chloride can reduce or alter
immunostaining, but it is not related to the pH of this solution.
In conclusion, the effects of calcium bound to tissue are highly complex, for calcium-
induced molecular modification may diminish antibody-antigen recognition or enhance
this effect. It is known that the ability of some monoclonal antibodies to recognize their
corresponding epitopes is calcium-dependent under certain conditions. The presence of
citrate buffer is not necessary to restore antigenicity, provided an appropriate pH is pres-
ent. The effect of bound calcium is not the only factor responsible for antigen masking.
In addition, modification of a protein molecule also occurs due to crosslinking introduced
by formalin fixation, causing antigen masking. Calcium binding to protein molecules
influences the immunoreactivity of some epitopes, while others are not affected. On the
other hand, heat-induced hydrolysis of protein crosslinks is the primary mechanism
responsible for epitope unmasking. Possible mechanisms responsible for epitope retrieval
by heat treatment are summarized below.
In summary, reasoned arguments have been presented in support of several mecha-
nisms responsible alone or in combination for antigen retrieval by heating; denaturation
and hydrolysis, self-assembly of unfolded protein chains and the subsequent restoration of
antigenic sites, chelation of calcium complexes, and unfolding of protein structure by
metallic salts or urea solutions through dissociation of hydrogen bonds or through the loss
of diffusable blocking proteins (Macintyre, 2001). In this respect, the role of residual
paraffin in the sections is unclear.
Antigen Retrieval 123
Because heating in water alone did not improve immunostaining, it is concluded that epitope
retrieval is mediated not only by heat and rehydration but also by the presence of a chelating
agent such as EDTA. However, caution is warranted in using EDTA, which may adversely
affect cell morphology because it is a strong oxidant.
Theoretically, any method of heating should unmask epitopes. Although most antigens
can be detected after heat treatment, some may be destroyed, and others may remain
masked. The temperature, changes in temperature during heating, and the duration of heat-
ing critically influence antigen retrieval. Different tissues, fixatives, and durations of fixation
require specific temperatures and durations of heating. Therefore, pilot studies should be
carried out to determine optimal heating conditions.
Similar staining intensities are achieved by the following heating conditions irrespec-
tive of the heating method used: 100°C for l0min, 90°C for 30min, 80°C for 50 min, and
70°C for 10hr (Shi et al., 1995a). Heating at 100°C for l0min (two cycles of 5min each) is
recommended for retrieving most antigens, except those damaged by high temperature. In
the latter case, lower temperatures for extended durations can be used. Overfixed tissues
require high temperatures and/or extended durations of heating. In some cases, durations of
heating longer than 10 min on a full-power setting may cause background staining.
Repeating the boiling cycles is more effective than extending the boiling duration.
This can be accomplished by removing the slide jar from the microwave oven after each
run and placing the slides in a new jar containing the fresh retrieval fluid at room temper-
ature, followed by again placing the jar in the oven. Compared with high-power microwave
outputs, medium wattage (e.g., 450 W) may yield better sensitivity, probably due to optimal
thermal effects and hence optimal oscillation of dipolar molecules.
ADVANTAGES OF HEATING
immunoglobulin light-chain immunostaining was better after microwave heating than after
trypsin digestion (Ashton-Key et al., 1996). Deparaffmized sections were heated on full
power in a 750 W microwave oven for 22min, while others were treated with 0.1% trypsin
for 10 min at 37°C. Similarly, in human biopsy tonsil tissue stains better after
microwave heating than after trypsin treatment (Fig. 6.5). If the retrieval of an antigen type
is adversely affected by heating, protease digestion of sections is the optimal pretreatment.
However, the preservation of cell morphology is generally better in heat-treated than in
enzyme-treated sections, especially when extended durations of digestion are employed.
HEATING METHODS
Different heating systems, such as microwave ovens (Shi et al., 1991), pressure cookers
(Norton et al., 1994; Miller and Estran, 1995), microwave heating–pressure cookers
(Taylor et al., 1995), autoclaves (Bankfalvi et al., 1994), steamers (Taylor et al., 1995),
water baths (Kawai et al., 1994), and electric hot plates (von Wasielewski et al., 1994), in
combination with antigen epitope retrieval fluids, have been used with various degrees of
success. Although each of the methods has minor advantages and limitations, they yield a
fairly similar degree of antigen retrieval when appropriate heating conditions are provided.
All the processing conditions must be adjusted for a specific study. Such conditions may
differ from those most widely cited in the literature or recommended by the manufacturers.
The choice of the heating method also depends on equipment availability.
A recent comparative study of the following five heating methods using 21 antibodies
also demonstrated that they produce similar intensities of immunostaining of retrieved
antigens provided the heating durations are adjusted appropriately (Taylor et al., 1996b).
However, heating methods Nos. 2, 3, and 4 (given below) yield better results. Advantages
and minor limitations of the heating methods are listed below.
1. Microwave heating for 10 min, carried out in a standard, simple, inexpensive, and
widely available microwave oven, is the fastest procedure. Total time required (including
set up of preheating, actual retrieval process, and cool down) is 25 min. A limitation is pos-
sible boiling over, resulting in the loss of antigen retrieval fluid. Consequently the level of
the fluid must be checked every 5 min. If necessary, more fluid can be added after the first
5 min to avoid drying the tissue sections. If more fluid is needed, this is the result of boil-
ing over, not evaporation. To catch any boiled-over fluid, the slide jar should be placed
within a larger jar which contains deionized water. In addition, the presence of hot or cold
spots in the microwave oven is not uncommon when several isolated jars containing the
slides are placed at random in the oven, a practice that leads to reduced reproducibility.
This method is also difficult to standardize. The microwave oven (900 W, 2,450 MHz) is
set at maximum power for two cycles of 5 min each.
Step-by-Step Protocol
Determine the optimal pH of antigen retrieval solution for each antigen. Citrate buffer
(0.01 M) adjusted to pH 6.0 with HC1 is used widely. Determine the desired temperature
based on the type of tissue and antigen under study. For fatty tissues, 90°C is recommended;
adjust the duration of heating accordingly. Place slides in plastic Coplin jars containing the
126 Chapter 6
Antigen Retrieval 127
antigen retrieval solution in the center of the rotary plate in the microwave oven to ensure
uniform heating of slides. Cover the jars with loose-fitting caps. Turn on the microwave
oven and check the temperature of the retrieval solution with a temperature probe. Use a
maximum power setting of 7–10.
Start timing the antigen retrieval duration when the retrieval solution begins to boil.
As an average, a total retrieval duration of 10 min, divided into two 5-min cycles with an inter-
val of 1 min between cycles to check the solution level in the jars, is recommended. If needed,
fresh retrieval solution from an adjacent jar can be added to the jars containing the slides.
Alternatively, distilled water can be used to replenish the retrieval solution. The objective is
to keep the slides fully immersed in the solution before restarting the oven. Alternatively, the
duration can be based on previously determined time for sections known to contain the anti-
gen under study. Remove jars from the oven, and cool the sections for 20 min at room tem-
perature. The slides are ready for immunostaining after being rinsed in 0.5 M PBS (pH 7.4)
for 5 min. Do not reuse the antigen retrieval solution. Some of the above-mentioned steps are
automatically controlled by the H2550 Laboratory Microwave Processor.
2. Microwave heating for 20 min is the same as Method 1 except that the heating is
employed four times for 5 min each. Total time required is 35 min. Improved immunos-
taining of many types of antigens can be achieved by extending the heating time. The pro-
cedure requires attention for 20 min to check the fluid level, and occurrence of hot or cold
spots may complicate the procedure.
3. Pressure cooking. Although microwave heating is widely used for antigen retrieval,
this system does not raise the temperature of an aqueous buffer above 100°C, even though
this temperature is reached rapidly. In contrast, an advantage of the pressure cooker is that,
if required, temperatures of 115°C or higher (superheating) can be achieved. Other advan-
tages of heating in a pressure cooker include short duration of heating, better repro-
ducibility of results with large batches of slides, the ability to use metal slide racks, and
economy of time and equipment cost (Norton et al., 1994).
Step-by-Step Protocol
Fill to approximately one-third capacity of a domestic pressure cooker (103kPa/15
psi) with 0.1 mM citrate buffer (pH 6.0). Bring the buffer to a boil using an electric hot
plate, without sealing the lid. Quickly place metal racks containing rehydrated section-
mounted glass slides into boiling retrieval buffer, and seal the pressure cooker. Bring the
cooker to full pressure. Start timing when the pressure indicator valve reaches the maximum
(~4 min). The optimal duration of pressurized boiling is 1–2 min. Depressurize the cooker
and cool it under running tap water. Remove the lid, and add cold tap water to replace the
hot retrieval buffer. A duration of 15–20 min is required to cool the cooker. Wash the slides
in several changes of 0.05 M PBS (pH 7.4) prior to immunostaining. At no time during this
processing are the slides allowed to dry out. The pressurized boiling (120–122°C) longer
than ~2min will progressively degrade the cell morphology.
4. Pressure Cooker–Microwave heating
The pressure cooker–microwave heating method is simpler than the autoclave proce-
dure and more efficient than microwave heating alone. The pressure cooker does not
require checking the level of the antigen retrieval solution during heating in the microwave
oven, and a large number of slides can be loaded simultaneously. In addition, the pressure
128 Chapter 6
cooker does not develop cold spots because it is a larger container. The limitation is that a
slightly longer duration (~45 min) is required than that for microwave heating alone.
Step-by-Step Protocol
Place slides in three plastic staining jars, each containing 24 slides and antigen retrieval
solution, and transfer them into a plastic pressure cooker (Nordieware, Minneapolis, MN)
filled with 600 ml of distilled water; this amount of water is one-half the capacity of the
cooker. Make sure that the jars stand stably in the water (Taylor et al., 1996b). Transfer
the pressure cooker into a microwave oven (model R-4A46) which is equipped to switch
one power level setting to another automatically. Place the cooker in the center of the
microwave oven. Set the oven at maximum power (900 W, 2,450 MHz) for 15 min to boil
the water, then switched to a 40% power setting for an additional 15 min to maintain mild
boiling (simmering). Remove the cooker from the oven, and allow it to cool for 15 min.
5. Autoclave heating, like pressure cooking, provides superheating at temperatures
higher than 100°C. Hydrated autoclaving eliminates the need to adjust the volume of the
antigen retrieval solution. Other advantages include the use of larger volumes of the
retrieval solution, which gives a uniform heating pattern and allows the heating of a large
number of slides in a single batch. In this method high-intensity immunostaining is
achieved, and the cold spots are absent. Hydrated autoclaving of slides, even in deionized
water, is thought to be more effective than either microwave or water bath heating (Shin et al.,
1991). It is known that heat denaturation of antigens is effective when the protein is
hydrated, whereas dehydrated protein is extremely resistant to heat denaturation. Minor
limitations are that the autoclave is expensive and may not be available in some small lab-
oratories. Also, the total time required to complete heating is ~45 min. Care should be
taken in handling owing to the pressure in the autoclave. The results of autoclave antigen
retrieval are shown in Figure 6.6 (Plate 3C, D, E).
Step-by-Step Protocol
Place slides in Coplin jars containing antigen retrieval solution that has been previously
heated at 80°C. Set the jars in the center of a stainless steel autoclave equipped with a 1,850
W heating filament. Tightly close the door of the autoclave as required by the instructions,
and heat at 120°C for 10 min at 15 psi. Cool down with running tap water for 20–30 min, then
rinse the sections with 0.05 M PBS at room temperature and immunostain.
6. Steam heating for 20 min over boiling water. Total time required is 35 min. This
method has the advantage that loading and unloading of slides into various carriers for auto-
mated use is not required. It needs relatively small amounts of the antigen retrieval fluid, does
not have cold spots, and is inexpensive. This approach is well suited to process a large num-
ber of slides simultaneously and thus saves considerable amount of time. Although originally
designed for autostaining, it can be used manually. A minor limitation is that preheated steam
is needed. Slides are set into the TechMate slide holder (Biotek, Santa Barbara, CA), with
antigen retrieval fluid in the capillary gap, and are heated by steaming over boiling water.
In contrast to microwave heating, steam treatment heats slides slowly to a uniform tem-
perature. This avoids boiling the antigen retrieval fluid and minimizes section detachment
from slides. Steam heat used in combination with EDTA and protease digestion has been
Antigen Retrieval 129
Step-by-Step Protocol
Sections mounted on a glass slide are placed in a beaker containing 1,000 ml of 0.2
M citrate buffer (pH 6.0) and heated on a hot plate (Corning, Utica, NY) for l0min at
100°C, and then allowed to cool at room temperature for 20min.
8. Equally good results, if not better in some cases, can be obtained with overnight
treatment of tissue sections in Tris buffer (pH 9.0) in a conventional oven at 70–80°C
130 Chapter 6
(Koopal et al., 1998). This method has been successfully used for retrieving antigens such
as estrogen and bcl-2 in cervix tissue and lymph node, respectively. Hot oven heating can
also be tried in a humidified chamber.
9. Another antigen retrieval method is hot water bath heating at 90°C for 120min.
Total time required is about It is simple and inexpensive, processes a large number
of slides each time, and does not require replenishment of the antigen retrieval fluid. The
method has been successfully used for the immunostaining of p53 and proliferating cell
nuclear antigens (PCNA) (Kawai et al., 1994). For p53 and PCNA retrieval, 0.01 M PBS
(pH 7.2) and 0.01 M citrate buffer (pH 6.0), respectively, are recommended (Fig. 6.7). The
use of this method is limited since it takes much longer time to complete. Hot oven heating
in a humidified chamber can also be tried.
The effects of microwave heating on the tissue sections that result in epitope retrieval
are exceedingly complex. A full understanding of the actions of microwaves at the molecular
level to facilitate epitope retrieval is lacking. At least two mechanisms need to be consi-
dered: heat and kinetic energy of the oscillating electromagnetic field. Both possibilities
are discussed below.
The most commonly accepted point of view is that heat is responsible for unmasking
the epitopes. In fact, Battifora (1996) has introduced the phrase heat-induced epitope
retrieval (HIER). Heating at 100°C is a powerful treatment that can unmask hidden, buried,
or crosslinked epitopes. Heat can be provided not only by a microwave oven, but also by
an autoclave, a pressure cooker, steam, or a hot plate. A consensus on which method of
heating is most effective in the retrieval of all types of epitopes is lacking. Therefore, some
Antigen Retrieval 131
factor or factors in addition to heat also become relevant. It is also known that treatments
other than heat can also unmask epitopes. Such treatments include enzyme digestion and
exposure to detergents.
The aforementioned observations indicate that heat is not the only mechanism respon-
sible for epitope retrieval. Therefore, the question arises, is the heat in the microwave oven
the only factor or the primary factor that facilitates epitope retrieval? Is it possible that in
addition to heat, kinetic energy plays a part in epitope unmasking (personal communica-
tion, A. S.-Y. Leong)? It is known that the microwave electromagnetic field causes polar
molecules in the tissue to oscillate at a rate of 2.45 billion cycles per second, enough to
disrupt protein crosslinking and unmask hidden epitopes. In addition, the fact that ultra-
sound treatment, which generates heat for an exceedingly short duration, also unmasks
epitopes, suggests that factors other than heat may also be important in explaining the
phenomenon of epitope retrieval in a microwave oven. The following explanation may
further understanding of the release of thermal energy and heat in a microwave oven.
Microwave energy is a nonionizing radiation (frequency, 300–300,000 MHz) that
causes molecular motion by migration of ions and rotation of dipoles. Dipole rotation
refers to the alignment, due to the electric field, of molecules that have either permanent
or induced dipole moments in both the solvent and specimens. As the field intensity
decreases, thermal disorder is restored, which results in thermal energy being released.
At 2,450 MHz (the frequency used in commercial systems), the alignment of the molecules
followed by return to disorder occurs times per second, resulting in rapid heating.
However, the absorption of microwave energy and its release as heat are strongly depen-
dent on the relative dielectric constant (relative permittivity) and the dipolar status of the
medium. The relative permittivity is the following ratio: material dielectric constant:
vaccum dielectric constant. The greater the relative dielectric constant, the more thermal
energy released, and the more rapid the heating for a given frequency (Camel, 2001).
Due to the particular effects of the microwaves on matter (namely dipole rotation and
ionic conductance), heating of the section, including its core, occurs instantaneously,
resulting in rapid breakdown of protein crosslinkages. Furthermore, the extraction and
recovery of a solute from a solid matrix with microwave heating is routinely obtained in
the field of analytical chemistry (Camel, 2001). However, a definite, full explanation of the
effects of microwave heating on the molecular aspect of antigen retrieval is awaited.
The duration of microwave heating to retrieve epitopes depends on the type of con-
centration of the aldehyde used for fixation, duration of fixation, and the temperature in
the microwave oven. The higher the concentration of the fixative and the longer the dura-
tion of fixation, the higher the temperature and the longer the duration of microwave heat-
ing required for epitope retrieval. The oven temperature is controlled using the temperature
probe of the oven and is checked with a thermometer. In a microwave oven with 720 W
power, the boiling point for the epitope retrieval fluid in the Coplin jar is
reached in 140–145 sec (Shi et al., 1994). The time it takes to reach a temperature of 55°C
is ~76sec. At 720 W, 5–10 min heating time is recommended, which can be divided into
two 5-min cycles with an interval of 1 min between cycles to check on the fluid level in
132 Chapter 6
plastic or glass jars. If necessary, more fluid at the same pH can be added after the first 5 min
to avoid drying the tissue sections. The jars can be covered with perforated cling film to
minimize evaporation.
Alternatively, 1 hr at 55°C or 30–120 min at 90°C (not boiling) can be used but is not
preferred. With long durations (24 hr–3 years) of fixation with formaldehyde, 20 min expo-
sure to heating at 100°C is recommended (Fig. 6.8) (von Wasielewski et al., 1994). A thor-
ough washing of slides after microwave heating in the presence of epitope retrieval fluid
and before incubation is essential to avoid background staining.
Although microwave heating, pressure cooking, wet autoclaving, and steaming of tissue
sections yield satisfactory antigen retrieval results, comparative studies indicate that pressure
cooking or pressure cooking in combination with microwave heating produces more uni-
form, efficient, consistent, and rapid immunostaining in some cases (Fig. 6.9). Pressure
cooking with or without microwave heating provides temperatures higher than 100°C (super-
heating). Such temperatures can be obtained with the high-pressure microwave processor
MicroMED URM (Sorisole, B G; Bergamo, Italy) (Suurmeijer and Boon, 1999). This appa-
ratus provides controlled superheating under high pressure in the microwave processor.
This processor has a maximal power output of 1,000W. The duration, temperature,
and pressure can be adjusted with a touch screen personal computer. Microwave power and
pressure are controlled through software. The pressure is regulated as a function of tem-
perature, which facilitates heating of the antigen retrieval solution at a constant tempera-
ture higher than 100°C without bubbling. A glass dome designed to withstand pressure
conditions rotates within the microwave cavity. The dome is provided with an automatic
raising and lowering mechanism controlled by the personal computer. A fiberoptic sensor
monitors the temperature of the antigen retrieval solution within the dome. The pressure in
the glass dome is between 1,900 and 2,000 mbar.
To obtain antigen retrieval, a plastic jar containing 250ml of 0.01 M citrate buffer
(pH 6.0) is centrally placed in the dome within the microwave cavity. Different tempera-
tures (ranging from 90–115°C), durations of heating (1–15 min), and pH values (2–10) can
be tested to determine optimal parameters for retrieving a given antigen. For example, opti-
mal immunostaining of Ki-67 antigen in malignant tumors using MIB-1 antibody was
achieved at 115°C for 10 min at pH 6.0 (0.01 M sodium citrate buffer) (Suurmeijer and
Boon, 1999). To my knowledge the use of this processor has not been reported by any
other laboratory, perhaps because of its high price.
Heating treatment is one of the most important factors influencing the effectiveness
of antigen retrieval on tissue sections. The heating of sections of the formalin-fixed and
paraffin-embedded tissues at a high temperature (boiling) for 10–20 min is extensively
used for retrieving many types of antigens. A variation of this method consists of heating
at high temperature, followed by heating at moderately low temperature.
Antigen Retrieval 133
134 Chapter 6
Microwave heating accelerates the process of staining for light and electron
microscopy, although this advantage is more useful for light microscopy because conven-
tional staining is quite rapid for electron microscopy. Under microwave heating, the
charged dye ions as well as the polar molecules and ions of the solvent, including water,
are excited (Kok and Boon, 1990). The molecular movement generated by microwave
heating may accelerate chemical reactions up to 1,200 times. The result is that heating
speeds up diffusion of stains into the thick-tissue sections and their subsequent reaction
and binding with the substrate.
Generally, compared with conventional staining, staining in the microwave oven
requires a much shorter staining duration and results in more intense staining, better con-
trast, and less nonspecific staining. In fact, the hours required for many conventional stain-
ing methods for light microscopy can be shortened into minutes. Several examples are
listed below. One example is the Grimelius method for staining neuroendocrine granules
in various tissues and tumors for light microscopy. The conventional procedure is com-
pleted in 3 hr, whereas the microwave method is accomplished in 3 min (Hopwood, 1992).
Also, staining of melanin can be carried out with colloidal silver nitrate in 45 sec under
microwave heating (Leong and Gilham, 1989a). Microwave heating is also effective in
reducing the tissue staining time from 70min to 15min for localizing acid and neutral
mucins with a modification of alcian blue periodic acid–Schiff stain (Matthews and Kelly,
1989). Microwave heat–stimulated staining of the brain tissue with the Rio-Hortego silver
impregnation technique can be completed within 24 hr instead of the 7 days required by
the conventional method (Marani et al., 1987). Satisfactory silver impregnation of cell
bodies, axons and their terminals, and dendrites and their spines is obtained.
Another example is the application of the Jones-Marres silver method for rapid stain-
ing of fungi in the brain tissue of immunocompromised patients (Boon et al., 1998).
This procedure can be carried out using the MicroMED BASIC microwave lab station
(Milestone, s.r.l., 24010 Sorisole, Italy). The lab station has software for reliable control of
power, time, and temperature using infrared temperature control for no-touch temperature
determination. It also has a 360-degree rotation carousel (no hot spots) and produces print-
outs of the temperature and power levels used during various microwave steps.
Microwave heat can also be applied for rapid staining of frozen sections. Frozen-
section diagnosis plays an important role in the evaluation of the operability of the patient
and in the examination of resection margins. The preparation for diagnosis, for example of
signet-ring cell carcinoma in the peritoneum, can be accomplished in as brief a time as 30 sec
by the modified periodic acid–Schiff’s (PAS) reaction facilitated by microwave heating
Antigen Retrieval 137
(Dworak and Wittekind, 1992). This protocol can demonstrate even a small number of mucin-
containing tumor cells surrounded by fibrous tissue in frozen sections (Fig. 6.10/Plate 3F).
Microwave heat is also effective in staining SDS-polyacrylamide gels with
Coomassie blue for visualizing as little as 5 ng of protein against a light blue background
of the gel (Wong et al., 2000). This protocol is one of the most powerful methods in mole-
cular biology for visualizing proteins.
Rapid staining of frozen sections of human brain tissue that has been stored in 10%
formalin (4% formaldehyde) for up to 10 years has also been reported (Feirabend and
Ploeger, 1991). In this study, rapid staining was obtained in the microwave oven by using
classic neuroanatomical staining methods such as Klüver-Barrera stain; originally Luxol
fast blue step required up to 24 hr, whereas in the microwave oven this step needed only
15–60 min. Another application of microwave heating is rapid staining of plant tissues with
dyes. For example, Safranin O can stain plant tissues in 45 min at 60°C in a microwave
oven instead of the conventional 48 hr at room temperature (Schichnes et al., 1999).
Microwave heat–assisted rapid fixation and double staining of the mouse fetal skele-
ton has also been carried out (Ilgaz et al., 1998). The staining was accomplished with a
mixture of alcian blue and alizarin red S in 23 min in the microwave oven instead of 4 days
at room temperature. The cartilage and bone are stained distinctly.
Most staining methods require optimal temperatures and durations of staining.
The optimal temperatures for most nonmetallic stains is 55–60°C, while for metallic stains
it is 75–80°C (Suurmeijer et al., 1990). Some specific examples are given below: the
Romanowsky-Giemsa method and the alcian blue technique at 55°C (Horobin and Boon,
1988), the Southgate mucicarmine procedure at 60°C, the Grimelius protocol at 75°C, the
Grocott, Jones, and Fontana-Masson methods at 80°C (Kok and Boon, 1990), and the gold
chloride (0.02%) at 74–98°C (Noyan et al., 2000). Some dye solutions, such as oil red O,
can be used at boiling temperature.
138 Chapter 6
Table 6.1 indicates microwave power levels, the stain used, and the required temper-
ature. Table 6.2 shows significantly reduced duration of staining under microwave heating
compared with conventional staining conditions. However, many brands of microwave
ovens are in use, and all vary in their performance, even at the same power level.
Therefore, optimal stain concentration, temperature of staining, and durations of staining
and rinsing will have to be determined for each type of new study. Also, care is required
in interpreting the staining results because high temperatures tend to produce staining arti-
facts. Autostainers for histochemistry are available from the following sources: Leica
Autostainer XL, Leica Instruments GmbH, Nussloch, Germany; Oticmax Rapid Microwave
Histoprocessor Inc., 160 Shelton Road, Monroe, CT 06468.
Using the horseradish peroxidase method and microwave heating, Ichihara et al.
(1989) were also able to immunostain frozen sections for the intraoperative diagnosis of
pancreatic cancer in 30min. Only four different antibodies were tested in this study.
A shorter duration of 10min has also been used for immunostaining frozen sections with
the EPOS system (Chilosi et al., 1994). The EPOS procedure is based on the chemical
linking of primary antibodies and horseradish peroxidase to an inert polymer complex
(dextran) (Bisgaad et al., 1993). This methodology has been employed for immunostaining
of Ki-67, PCNA, cytokeratin, and leukocyte common antigens (Tsutsumi et al., 1995;
Richter et al., 1999). The limitation of the standard EPOS system is that the primary anti-
bodies are labeled and thus are commercially available only for limited range of antigens.
In contrast to the EPOS system, a modification of the highly sensitive two-step
irnmunohistochemical EnVision system allows the detection of a broad spectrum of anti-
gens in frozen sections in less than 13 min (Kämmerer et al., 2001). In this study 38 out of
45 antibodies tested showed specific staining. In fact, the modified EnVision procedure
allows the use of any suitable primary antibody, preferably monoclonal antibodies. Like
the EPOS system, EnVision employs a dextran polymer coupled to horseradish peroxidase
molecules for detection. No attempt was made to block endogenous peroxidase, nor was
any antigen retrieval pretreatment used. Because of the very short incubation durations, a
humid chamber is not required to avoid evaporation of immunoreagents.
A minor disadvantage of the modified EnVision system is that it requires primary
antibody concentrations four- to tenfold higher than those used in the conventional
immunohistochemical procedures. Another limitation of this modified method is that only
two slides with two sections each can be processed at any one time.
The sections are rinsed in TBS, and then exposed to DAB+ chromogen (Dako) for a
few minutes as the substrate for the EnVision-HRP-enzyme. The sections are washed by
shaking the slide rapidly under tap water for 10 sec. The excess fluid is removed from the
slide with a paper towel. The slide is dipped in distilled water, counterstained with Meyer’s
hematoxylin for 15 sec, and then rinsed in hot tap water at 42°C for 30 sec.
Antigen Retrieval 141
The causes of most hazards encountered in using a microwave oven are straightforward
and can be avoided by taking necessary precautions. Higher-power settings and longer
durations of heating than optimal for a given study should be avoided. Because overheat-
ing is not uncommon, the time setting should be checked. The fluid contents of the con-
tainer heat faster than the container. In fact, the fluid contents of the container heat so fast
that the container can still be cool (Marani, 1998). Even after the container has been
removed from the oven, it will become hotter for a period of time. Changes in the size,
shape, and nature of the container and its position in the microwave oven significantly
change the temperature of the container fluid. Furthermore, changes of these factors will
change the temperature of the container fluid even if the volume of the container contents
remains unchanged.
Overheating the microwave oven tends to result in boiling or excessively rapid evap-
oration of fluids such as ethanol used for dehydration, formaldehyde employed for fixa-
tion, and the antigen retrieval fluid. As a result, flammable and/or toxic materials are
released in the microwave oven. Even without overheating, vapors are produced because
containers are kept open in the oven to prevent pressurization. Transparent microwave con-
tainers should be used, fluid volumes should be ~100ml. Microwave ovens with attached
efficient extractor fans are commercially available, as are microwave ovens with tempera-
ture probes. To avoid possible exposure to toxic vapors, the face should be turned away
when the oven door is opened (Horobin and Fleming, 1990). The oven door should not be
opened or closed to turn the microwave power on and off.
When using Pelco 3440 MAX laboratory microwave oven (Ted Pella, Redding, CA),
areas of high microwave flux should be checked, using a Pelco 3,614 microwave bulb
array (Ted Pella) (Fig. 6.12). Specimens should not be placed in areas indicated by illu-
minated bulbs. Vials containing the specimens should be placed in a water bath (50 ml) that
has been preheated to the required temperature. The temperature should be regulated by
placing a microwave temperature probe into a vial of the same solution that is present in
the specimen vial. The built-in temperature probe displays the temperature on the oven
front panel. The wire that attaches the probe to the oven should be submerged in the water
to decrease the antennae effect (Schichnes et al., 1999). An additional 400ml of static
water load should be placed in the oven at an optimal position determined with the
microwave bulb array. This water is changed between every step.
Pelco BioWave microsystem is the latest advancement in microwave heating tech-
nology. It is equipped with vacuum cycling (down to 1 torr), variable wattages, and a pre-
cision temperature probe; it can accommodate Pelco coldspot connected to the Pelco load
cooler and thus eliminate hot and cold spots during processing. The system can be used for
both light and electron microscopy. The vacuum chamber is most helpful during fixation
and infiltration of tissue specimens.
The following specific steps must be taken while using a microwave oven for antigen
retrieval (Marani, 1998).
1. Test microwave leakage with a microwave detector with a low sensitivity range.
2. Place the oven in an efficient fumehood.
3. Wear gloves while using your hands inside an oven.
142 Chapter 6
In spite of the overwhelming advantages of microwave heating, some real and possi-
ble limitations are described. Background staining may occur with some antibodies, partic-
ularly when the heating is prolonged. This problem can be avoided by determining the
optimal temperature and time of heating by trial and error. Although antigen specificity
Antigen Retrieval 143
for the monoclonal antibody is maintained after microwave treatment, the possibility of
altered immunostaining should not be disregarded whenever new or previously untested
antibodies are used. The results of such studies should be compared with those obtained
using frozen section immunohistochemistry.
In some cases, microwave heating may damage nuclear morphological details,
including mitotic figures. This problem may lead to difficulty in identifying cells accu-
rately, which is important in diagnostic studies. Both mitotic figures and morphology, for
example, are important in distinguishing a malignant lymphoid infiltrate within a mixed
cell population (Hunt et al., 1996). In such studies, it is desirable to use a heating method
other than microwaves and accept slightly lower immunostaining enhancement.
Although the exact knowledge of molecular changes responsible for impairment of
nuclear morphology caused by microwave heating is lacking, it may be possible that this
treatment causes some structural damage to intracellular macromolecules, resulting in an
increase in the number of osmotically active moieties within the nuclear compartment,
thus attracting water and causing nuclear swelling (Hunt et al., 1996). Such swelling would
blur mitotic figures, leading to a less accurate count of them.
Some other limitations and their avoidance are described below. With violent boiling
and extensive evaporation of the retrieval fluid in which the sections are immersed, the
sections should be monitored to avoid drying and damage. To obviate this problem,
microwave heating must be performed in repeated bursts; the plastic jars must be refilled
following each cycle or a large reservoir of retrieval fluid or distilled water must be placed
in the oven. To avoid inconsistent results, plastic jars containing the slides should always
be placed every time in the same location in the microwave oven. The number of slides and
jars should be constant every time a microwave oven is used, even when this entails insert-
ing blank slides into the jar (Gown et al., 1993). Tissue sections should be placed toward
one end of the slide (lower side of the slide while placing it in the jar) to ensure continu-
ous immersion in the epitope retrieval fluid during microwave heating.
Uneven distribution of microwaves within the oven results in hot and cold spots (see
pages 102–103). This problem can be avoided by placing a 500-ml water load in the rear
of the oven and by using a turntable during the process of heating (Panasonic model NN
5652, 800 W). Only a limited number of slides can be accommodated in the microwave
oven. Tissue section detachment from the glass slide may occur during heating, especially
with tissues containing prominent fibrous elements (Cuevas et al., 1994). If this problem
is encountered, the surface of the slide can be made adhesive for sections by coating it
with poly-L-lysine or 3-aminopropyl-triethoxysilane or, still better, by using electrically
charged glass slides. Another problem is that microwave ovens have the inherent disad-
vantage of decreased power generation with use. Thus, no two ovens in use will have the
same heating characteristics. This limitation is an obstacle in standardizing antigen
unmasking methods.
Microwave heating in some cases is not desirable. This method, for example, causes
complete loss of estrogen receptor immunoreactivity, even when monoclonal antibody
H222 is used (Gown et al., 1993; Leong, 1996). In such cases, alternate procedures, such
as enzyme digestion alone or followed by microwave heating, can be used. Similarly,
another steroid hormone receptor androgen shows stronger immunostaining with auto-
claving than that using microwave heating (see Fig. 6.13) (Ehara et al., 1996). Another
example is insulin, which shows diminished immunoreactivity after microwaving in citrate
144 Chapter 6
buffer (pH 6.0) (Tornehave et al., 2000). In such cases alternative antigen retrieval fluids
and heating methods are required.
Contrary to some reports, microwave heating in some cases does not abolish con-
taminating immunostaining during the consecutive detection of two or more types of anti-
gens within the same section. This problem is especially common when double
immunolabeling with antibodies of the same species and isotope is used. Recently it was
demonstrated that microwave heating did not completely abolish contaminating staining
when cytoplasmic and nuclear antigens in proliferating cells were labeled in cryostat and
paraffin sections, with primary monoclonal antibodies from the same species and the same
isotope being used (Bauer et al., 2001). However, such contaminating staining can be
avoided in some cases with the use of microwave heating. Lan et al. (1995) have reported
blocking of antibody cross-reactivity in multiple immunoenzyme staining and retrieving
antigens with microwave heating.
In summary, although the microwave heating method is highly effective for detecting
a large number of tissue-bound antigens which otherwise may remain masked, primarily
due to fixation with formaldehyde, certain antigens show reduced immunoreactivity
following microwave heating. It should also be noted that epitope retrieval with
microwave heating or other methods can unmask cross-reactivities that can be very diffi-
cult to deal with. Therefore, the use of retrieval methods for immunostaining creates the
necessity for increased vigilance in the selection and interpretation of controls, both
negative and positive.
Antigen Retrieval 145
Procedure 1
Tissues are fixed with formalin for 18 hr to 4 weeks and then embedded in paraffin.
Sections are mounted onto superfrost or poly-L-lysine–coated glass slides, dried
in an oven for 1 hr at 60°C, and deparaffinized with three changes of xylene. This is followed
by rehydration through a series of descending concentrations of ethanol. The slides are placed
146 Chapter 6
in plastic Coplin jars containing 0.01 M sodium citrate buffer (pH 6.0), which are heated in an
autoclave for 5–10 min at 120°C. The slides are allowed to cool down to room temperature for
20–30 min and then briefly rinsed in 0.05 M Tris-HCl buffer (pH 7.4) or 0.1 M PBS.
Blocking of endogenous peroxidase activity is accomplished by immersing the sec-
tions for 30 min in a solution of 0.3% in distilled water, and then rinsing in PBS. If
needed, background staining can be blocked by treating the sections for 10–30 min at room
temperature with normal serum from the species supplying the second antibody at a dilu-
tion of 1:5 to 1:20 in PBS. The sections are incubated overnight in a humidified chamber
at 4°C in the primary antibody at an appropriate dilution. They are rinsed three times for
5 min each in PBS, further processed by using avidin-biotin complex (Vectastin, Vector
Labs, Burlingame, CA), followed by DAB as the chromogen. Counterstaining of the nuclei
is accomplished with hematoxylin or methyl green. As a negative control, irrelevant anti-
body UPC10 (Cappell, Organon Teknika, West Chester, PA) can be used or primary
antibody can be omitted. The sections are mounted in an appropriate mountant.
Procedure 2
ULTRASOUND TREATMENT
The main part of the converter is a lead zirconate titanate electrostrictive element that
expands and contracts when subjected to alternating voltage (Portiansky and Gimeno,
1996). The converter vibrates in a longitudinal direction and conveys this motion to the
horn tip immersed in the solution, resulting in the implosion of microscopic cavities in the
solution. The implosion causes the molecules in the solution to become exceedingly
agitated. This phenomenon is explained below.
Some information is available on the mechanisms responsible for the direct or indirect
effects exerted by ultrasound on antigen retrieval. Considerable heat is generated during
ultrasound exposure, but the heat dissipates very quickly. Very rapid heat loss has misled
some workers to state that “ultrasound generates a mild increment in temperature”
(Portiansky and Gimeno, 1996).
Ultrasound waves consist of cycles of compression and expansion. Compression
cycles exert a positive pressure on the liquid, pushing the molecules together, whereas
expansion cycles exert a negative pressure, pulling the molecules away from each other.
The tensile strength of solutions is reduced by gas trapped in the crevices of small solid
particles in the solution. When a gas-filled crevice is exposed to a negative pressure cycle
from a sound wave, the reduced pressure makes the gas in the crevice expand until a small
bubble is released into the solution, initiating cavitation. A negative pressure of only a few
atmospheres will form bubbles. The bubbles ( in diameter) implode violently in
less than a microsecond, intensely heating their contents (Suslick, 1989). Thus, during the
expansion cycle a sound wave of sufficient intensity can generate cavities in the solution.
Ultrasound treatment causes enormous molecular agitation (turbulence), heat, and
pressure of imploding cavities. Such agitation not only initiates but also accelerates both
biochemical and physical reactions. In other words, effects of ultrasound involve processes
that create, enlarge, and implode gaseous and vaporous cavities in a solution. The implo-
sion of cavities also sends shock waves through the solution. This extreme condition gen-
erated by cavitation can induce reactivity between cellular proteins and the antigen
retrieval solution (e.g., sodium citrate). Mechanical vibrations and high temperatures may
extract tissue-bound calcium ions, accelerating the chelating effect of citrate. This sugges-
tion is reinforced by the evidence that ultrasound hastens calcium chelation and bone
decalcification (Thorpe et al., 1972; Page et al., 1990). Chelation of calcium may result in
epitope retrieval (Morgan et al., 1994).
It is known that ultrasound can break or disrupt cells and tissues. Mechanical vibra-
tions generated by ultrasound can induce structural changes in the tissue sections, break-
ing the formalin-introduced protein crosslinks and thus facilitating the accessibility of
antigens to antibodies. Ultrasound can also unfold or “crack” protein molecules into
smaller fragments, exposing the epitopes.
The effectiveness of ultrasound treatment in epitope retrieval has been compared with
that achieved with microwave heating or pressure cooker alone (Portiansky and Gimeno,
1996). It was shown that ultrasound was more effective in immunostaining prostatic basal
cell structural cytokeratins. The capability of microwave heating for epitope retrieval has also
been compared with that of ultrasound in combination with microwave heating (Brynes et
al., 1997). The latter approach resulted in stronger immunostaining with lower nonspecific
background staining of cyclin Dl bcl-1 nucleoprotein in mantle cell lymphoma specimens.
It should be noted that raising the temperature beyond a certain level in the presence or
absence of ultrasound does not improve epitope retrieval and in addition results in excessive
148 Chapter 6
Procedure
Tissues are fixed with 10% formalin for 7–10 days and embedded in paraffin
(Portiansky and Gimeno, 1996). Sections about thick are mounted on glass slides
coated with poly-L-lysine and deparaffinized with xylene. They are incubated with 0.03%
methanolic hydrogen peroxide for 30 min to inhibit endogenous peroxidase activity.
Following dehydration with graded ethanol, they are rinsed in deionized water and then in
PBS. The glass slides containing these sections are vertically oriented in the lateral walls of
a 75×95-mm glass dish and completely covered with 10 mM citrate buffer (pH 6). The tip
of the cell is disrupted (Branson Ultrasonics model 250), set to continuous mode, and
immersed 3 cm in the citrate buffer in the center of the dish. After incubation in the primary
antibody (appropriately diluted), the avidin-biotin complex (ABC) is used as the detection
system. In control sections, the primary antibody is replaced with normal mouse serum.
NONHEATING METHODS
Detergents
Antigen retrieval using heat-based methods is not being widely used for cell cultures
and cryosections fixed with an aldehyde. The immunolabeling efficiency of such specimens
can be improved by using a chemical antigen retrieval protocol. This protocol consists of per-
meabilizing the specimens with Triton X-100, followed by treating with sodium dodecyl
sulfate (SDS). This permeabilization/denaturation treatment is applied after fixation and prior
to incubation with the primary antibody. SDS is the most commonly used denaturing agent
for gel electrophoresis. The application of SDS in epitope retrieval is based on the observa-
tion that after treatment with this reagent, protein bands appear in gel electrophoresis, but
Antigen Retrieval 149
such results with similar proteins without SDS treatment using immunocytochemistry are
not visible. Being a protein denaturant, SDS application may result in bands after staining
with Coomassie blue, but it itself is not a stain. Therefore it should not be referred to as a
positive or negative stain.
SDS disrupts noncovalent interactions between subunits of a protein, so if a protein
has two subunits, two bands will appear. In the absence of SDS, only one band will appear.
This reagent and mercaptoethanol reduce protein subunits that are disulfide bonded. This
property of SDS may be responsible for protein denaturation. It should be noted that SDS
also permeabilizes cells for antibody access to intracellular epitopes.
Triton X-100 and digitonin are also used to permeabilize the cell membrane allowing
antibody penetration. Triton X-100 permeabilization of formaldehyde-fixed cells allows
antibodies better access to their epitopes than does digitonin treatment. Digitonin or saponin
binds to cholesterol within membranes, creating digitonin-cholesterol complexes and pores
in the membrane. The pores are sufficiently large to allow antibody penetration. On the
other hand, Triton X-100 is a stronger detergent and dissolves most of the membrane lipids.
As a result this detergent increases the accessibility of antibodies to cell compartments that
are not permeabilized with digitonin (Hannah et al., 1998). A limitation of Triton X-100 is
that it may extract certain antigens even from fixed cells. Thus, false-negative staining of
antigens, especially membrane antigens, of the cells treated with a strong detergent can
occur because of antigen extraction. However, it should be noted that not all membranes of
formaldehyde-fixed cells are impermeable to antibodies without permeabilization.
The permeabilization/denaturation method has been successfully used for immunola-
beling of in human neutrophils and MRC-5 cells (Robinson and Vandré, 2001).
The method has also been effective in labeling MDCK cells in conjunction with indirect
immunofluorescence (Brown et al., 1996). It should be noted, however, that some type of
antigens remain masked, while other types may be adversely affected by SDS treatment.
Still other antigens (e.g., aquaporins and brush border gp330) remain unaffected by SDS
treatment (Brown et al., 1996). An example of an antigen whose staining is negatively
affected by SDS treatment is in the Golgi complex; this occurs with the anti-AE1 anion
exchanger antibody (Brown et al., 1996). Therefore, the usefulness of the SDS treatment
should be assessed in each case. Caution is also required to prevent drying out of the speci-
mens during incubation steps because they become hydrophobic with SDS treatment.
Procedures
Kidney tissue is fixed with paraformaldehyde-lysine-periodate by vascular perfusion
(Brown et al., 1996). Tissue slices are further fixed overnight at 4°C with the same fixative
and stored in PBS (pH 7.4) containing 0.02% sodium azide. They are placed in 30%
sucrose in PBS for at least 1 hr, and then surrounded by a drop of Tissue-Tek embedding
medium on a cryostat chuck before freezing by immersion in liquid nitrogen. Cryostat sec-
tions about thick are cut at a chamber temperature of –25°C, collected on Fisher
Superfrost Plus charged slides, and stored at –20°C until use.
The sections are brought to room temperature, and a wax pen (PAP pen, Kiyota
International) is used to trace a hydrophobic circle around each section. They are rehy-
drated by immersion in PBS for 5 min; most of the PBS is removed from the slide with a
tissue paper and the sections are then covered with drops of SDS solution (1% SDS in
150 Chapter 6
PBS). The drops are confined to areas where the sections are encompassed by the wax cir-
cles. After the slides have been treated horizontally for 5 min at room temperature, the slide
is immersed in PBS in a Coplin jar to remove the SDS. The control slide not exposed to
SDS is washed in a separate jar to avoid any contact with SDS. The slides are thoroughly
washed three times for 5 min each with PBS, completely removing the SDS; otherwise,
residual SDS will denature the antibodies subsequently applied to the sections.
While the slide is horizontal, excess PBS from the areas outside the wax circles is
removed. The sections become hydrophobic after SDS treatment, so care must be taken to
prevent them from drying. The aliquot of primary antibody should be already in the
pipette, so that it can be applied to the sections immediately after the residual PBS has
been removed. The sections can be incubated in the primary antibody for 1–2 hr at room
temperature, followed by two washes for 5 min each in high-salt PBS (containing 2.7%
NaCl instead of 0.9% NaCl). This PBS minimizes nonspecific binding of antibodies to the
tissue. After being washed for 5 min in normal PBS, the sections are incubated in the sec-
ondary antibody (goat antirabbit IgG conjugated to fluorescein isothiocyanate, FITC) for
1 hr. This is followed by washing in normal PBS, then mounting of sections in the medium
of choice (Fig. 6.14).
A second nonheating epitope retrieval method involves the use of sodium hydroxide-
methanol solution. This solution was used successfully for epitope retrieval in sections of
formalin-fixed, acid-decalcified human temporal bone embedded in celloidin (Shi et al.,
1991). This solution is prepared by adding 50–100 g of NaOH to 500 ml of methanol in a
brown bottle and mixing vigorously. The solution can be stored for 1–2 weeks at room
temperature; it is also available commercially (BioGenex, San Ramon, CA). The clear,
saturated solution is diluted 1:3 with methanol before use. A wider application of this
solution is awaited.
Another reagent used to unmask epitopes by denaturing antigens is guanidine
hydrochloride (GdnHCl) which is freely soluble in water and alcohol; its
Antigen Retrieval 151
aqueous solution has neutral pH. It was used for retrieving masked or hidden intracellular
protein in scrapie-infected cultured cells (Taraboulus et al., 1990). A modified version of
this protocol was employed for localizing different epitopes in BHK-21 cells fixed with
paraformaldehyde containing small amounts of glutaraldehyde (Peränen et al., 1993).
These epitopes were undetectable without denaturing the antigens with GdnHCl, using
immunofluorescence microscopy.
The advantage of denaturation of antigen complexes using GgnHCl is that it allows
the use of glutaraldehyde, a more potent protein cross-linker, which better preserves pro-
tein cell structure. This means that low concentrations of this dialdehyde do not interfere
with the epitope retrieval property of GgnHCl. The use of glutaraldehyde widens the appli-
cations of this denaturing agent. Both monoclonal and polyclonal antibodies can be used
in conjunction with GgnHCl. Guanidine hydrochloride is especially useful with antibodies
known to react only with denatured antigens. This reagent also permeabilizes cells.
A limitation is that GgnHCl tends to eliminate the antigenicity of certain intra-
cellular structures such as microtubules. However, microtubule loss can be prevented
by using low concentrations of glutaraldehyde during fixation with paraformaldehyde
(Peränen et al., 1993).
A variety of proteolytic predigestions have been employed for unmasking epitopes that
had become inaccessible as a result of crosslinking during aldehyde fixation. The digestive
treatments have been carried out most commonly with trypsin, pepsin, proteinase K,
or pronase (their concentrations are given later) prior to immunostaining. Detailed com-
parative studies on the effects of these four enzymes on epitope unmasking demonstrate
that while the results did not differ significantly among themselves, their effects did differ,
depending on the tissue and the antibody used (Hazelbag et al., 1995). Other factors affect-
ing such results include the duration of digestion, pH, temperature, and length of fixation.
The mechanism responsible for antigen retrieval by enzymatic digestion is break-
down of protein crosslinks formed during formalin fixation. It is likely that enzyme treat-
ment digests surface binding proteins, exposing the masked antigenic sites for antibody
binding. This idea is supported by evidence that the duration of enzymatic digestion
required for epitope retrieval is proportional to the length of formaldehyde fixation. It is
also known that overdigestion leads to damage, not only to cell morphology but also to
immunoreactivity.
Enzymatic digestion is preferred over microwave heating for antigen retrieval in a few
cases. Even multiple enzymatic digestion is required to retrieve certain antigens in a spe-
cific tissue. As an example, it has been reported that the monoclonal antibody RCC is most
effective in the staining of clear cell carcinomas and papillary carcinomas in renal neo-
plasms when sections are pretreated with a three-step enzymatic digestion method: 0.12%
trypsin in Tris-buffered saline (TBS), 0.01% pronase in TBS, and 0.1% pepsin in 0.1 N
HC1. Results were inconsistent with heat-induced epitope retrieval techniques. However,
trypsin is used most commonly, which catalyzes the hydrolysis of orginyl and lysyl pep-
tide bonds. Trypsin usually is used at a concentration of 0.1% in 0.05 M Tris/HCl buffer
(pH 7.8) containing 0.1% for 20–40min at 37°C. The addition of is essential
152 Chapter 6
for controlling digestion and reducing the production of occasional white flocculation
(Macintyre, 2001). Only freshly prepared solutions of these enzymes should be used, as
enzyme activity decreases with age. Also, these solutions should be prewarmed to the
required temperature to ensure consistent results.
Proteolysis does have certain limitations. Some antigens are susceptible to enzyme
digestion. In some cases insufficient unmasking can result in poor or false-negative results,
while excessive digestion may adversely affect cytomorphological features and cause
increased background staining and detachment of tissue sections from the slide. It is
known that proteolysis is a potent treatment. Because the cleavage of the protein molecule
by proteolytic enzymes is mostly nonspecific, these reagents may alter the epitopes.
In other words, peptide bond cleavage by these treatments is largely nonspecific. Therefore,
these procedures are not the preferred treatments. Nevertheless, enzymatic digestion is
useful for a limited panel of antibodies. If needed, enzymatic pretreatments can be applied
preceded by microwave heating (Dookhan et al., 1993).
Procedure
Slides with tissue sections are treated with 0.1% trypsin solution containing 0.1%
(pH 7.4) for 15 min at 37°C, with 0.4% pepsin solution containing 0.01 MHC1 for
20 min at 37°C, or with 0.025% pronase E solution containing 0.05 M Tris-HCl (pH 7.6)
for 15 min at the same temperature (Hazelbag et al., 1995). These are average concentra-
tions and durations, which should be adjusted according to the tissue and antigen type and
the duration of fixation. Prolonged fixation requires longer proteolysis to unmask the epi-
topes. Excessive proteolysis results in decreased immunostaining. If loss of the sections
during proteolysis is a problem, the slide can be coated with a 3% solution of casein white
glue and dried overnight before the sections are placed on it.
with PBS and then incubated overnight at 4°C in the MIB-I antibody (Immunotech,
Westbrook, ME), diluted 1:50 with PBS.
An automatic immunostainer (Cadenza, Shandon Scientific Inc., Pittsburgh, PA) can
be used to accomplish staining. The Supersensitive Streptavidin-AP detection kit
(BioGenex, San Ramon, CA) is used according to manufacturer’s directions. The final
color reaction is developed with a fast red substrate (BioGenex), followed by mild hema-
toxylin counterstaining to avoid masking weak immunostained nuclei. Slides are cover-
slipped with Crystal Mount (Biomedia Corporation, Foster City, CA).
The following recent comparative studies demonstrate that no single antigen retrieval
method is optimal for all types of antigens.
10. Ultrasound treatment was compared with microwave heating and pressure cooking;
the former treatment was claimed to be quantitatively and statistically superior
for the immunostaining of prostatic basal cell structural cytokeratins using mon-
oclonal antibody K8.12 (Portiansky and Gimeno, 1996).
11. Compared with trypsinization–microwave heating, microwave heating–
trypsinization demonstrated optimal immunostaining of Ki-67 using monoclonal
antibody MIB-1 (Szekeres et al., 1995).
12. Immunostaining using a panel of 21 antibodies was compared by employing
microwave heating, microwave–pressure cooking, autoclave, and steamer (Taylor
et al., 1996b). These methods yield similar intensities of staining provided the
durations of heating are appropriately adjusted.
13. Immunostaining of cytokeratin 18 in normal and neoplastic hepatocytes using
antibody CK 18 was compared by employing microwave heating (15 min), auto-
claving (10 min), pressurized boiling (1min), and simple boiling (15 min) in
10 mM citrate buffer (pH 6.0) (Xiao et al., 1996). No difference was found in the
degree of immunostaining with light and electron microscopy.
14. Compared to microwave heating, digestion with proteinase K for 2–4 min at
room temperature yielded better retrieval of cytokeratins in mouse tissues using
monoclonal antibodies (e.g., AE1, AE3) generated against human cytokeratins
(Martin et al., 2001).
15. Immunostaining of a number of proteins between microwave heating at 100°C of
20 min and boiling on a conventional hot plate. No difference was observed in the
results of the two methods (Varma et al., 1999).
16. Antigens bcl-2, CD3, and CD79a in tonsil tissue embedded in methyl methacrylate
show superior immunostaining with trypsin followed by superheating at 121°C in
a pressure cooker compared with that obtained with microwave heating only
(Hand and Church 1998).
17. Among the three antigen retrieval methods, hydrated autoclaving, microwave
heating, and simple heating, simple heating overnight at 60°C was most effective
for smooth muscle actin labeling (Igarashi et al., 1994).
18. More intense and widely distributed staining of cytokeratins was observed with
protease digestion than with microwave heating in benign lesions in the prostate
using mouse monoclonal antibody (Googe et al., 1997).
Chapter 7
Antigen Retrieval on
Resin Sections
155
156 Chapter 7
Polarbed 812 50 g
DDSA 32 g
MNA 21 g
DMP-30 2g
It is well established that formaldehyde reacts with amino groups on protein side chains
(Fig. 7.2), introducing mostly reversible protein crosslinks. Epoxy monomer reacts with the
Antigen Retrieval on Resin Sections 157
new hydroxyl groups introduced on the protein by formaldehyde; the epoxy molecules
thereby are copolymerized with the protein. In other words, formaldehyde functions as a link
between protein side groups and the epoxy monomer (Brorson et al., 1999). In contrast, such
a copolymerization does not occur between acrylic resins and tissue proteins. These resins
permeate the tissue without chemically binding to them. Accordingly, during thin section-
ing, the two resins cleave differently. In the case of acrylic resins, the surface of cleavage
tends to follow the path of least resistance; this path is the interface between the resin and
proteins. Thus, more epitopes without splitting are exposed at the surface of acrylic sections.
On the other hand, in the case of epoxy sections, the resistance in such interfaces is
not significantly less than that in tissue proteins, which results in the splitting of protein
158 Chapter 7
molecules. Also, the surface of epoxy sections is smoother than that of acrylic sections.
Consequently, fewer epitopes are exposed on the surface of the former sections. However,
epoxy resins are of value, being easier to cut and more stable under the electron beam and
better preserving the ultrastructure.
Antigen sites can be unmasked not only on thick and semithin resin sections for light
microscopy but also on thin resin sections for electron microscopy. Antigen retrieval at the
ultrastructural level has been accomplished on thin sections of epoxy resins (Stirling and
Graff, 1995; Röcken and Roessner, 1999) and LR White resin (Wilson et al., 1996;
Sormunen and Leong, 1998). Because epoxy and LR White resins are superior to some
other resins with respect to preserving the cellular details and other characteristics, antigen
retrieval methods using these two resins for electron microscopy are presented.
For electron microscopy, tissues can be fixed with a mixture of formaldehyde and
glutaraldehyde or with the latter only. Glutaraldehyde fixation better preserves cellular
details but strongly masks antigens. However, antigenic sites can be unmasked on epoxy
thin sections of glutaraldehyde-fixed tissues by exposing the sections to strong oxidizing
agents such as EDTA, hydrogen peroxide, sodium methoxide, or sodium metaperiodate.
These treatments also allow immunostaining of sections of postosmicated tissues by
removing osmium bonds. Moreover, such treatments temporarily minimize the hydropho-
bicity of epoxy section surface and may increase resistance to heavy metal poststaining
(Bendayan and Zollinger, 1983; Causton, 1985; Newman and Hobot, 1993).
The above-mentioned etching pretreatments are generally useful for epoxy sections
but not for acrylic (LR White) sections because unlike acrylic resins, epoxy resins form
covalent bonds with proteins. In other words, epoxy resins copolymerize with the tissue,
while acrylic resins surround the tissue components without becoming part of them.
Accordingly, epoxy resins strongly mask the proteins that become mostly inaccessible to
antibodies. Therefore, epoxy sections, especially of glutaraldehyde-fixed tissues, require
etching to unmask the antigens.
The surface of acrylic sections is rougher than that of epoxy sections. Moreover,
acrylic sections are less crosslinked and more hydrophilic than epoxy sections. As a result,
immunostaining reagents penetrate acrylic sections easily, facilitating antigen detection.
Exposure of acrylic sections to oxidizing agents worsen both the known instability of these
sections under the electron beam and the structural details.
To facilitate the access of antigens to antibodies, the protocol of embedding and etch-
ing given on page 159 is used (Crowley, 1997). The sections of this low-crosslinked
embedding medium are thought to allow easy penetration of aqueous immunostaining
fluids.
A saturated solution of sodium periodate is prepared by dissolving 1 g of this reagent
in 5 ml of distilled water and passing the solution through a pore filter. The grids
are wetted by floating them on drops of distilled water and then floated on drops of
the sodium periodate solution for ~15 min (this duration can be changed to obtain maxi-
mum immunoreactivity). The grids are thoroughly rinsed in distilled water and must not
be allowed to dry before immunostaining.
Antigen Retrieval on Resin Sections 159
Embedding Media:
Araldite 502 15ml
Eponate 12 25ml
DDSA 55ml
Dibutyl Phthalate 1%
DMP-30 1.5%
If background staining is a problem and standard rinsing with PBS fails to reduce
nonspecific staining, boosting the sodium chloride concentration from ~0.9% (150 mM)
to ~4.5% (750 mM) may help (Chiovetti, 1998). After this treatment, the grids must be
rinsed several times in standard PBS before further processing, so that the salt concentra-
tion is reduced to the range of physiological strength. As an example, the grid can be rinsed
five times for ~2 min each on drops of high-salt buffer, followed by two rinses for ~2 min
each on drops of standard salt buffer. This routine can be used after incubation in the
primary antibody or any other incubation (e.g., secondary antibody incubation, colloidal
gold) that is suspected of contributing to nonspecific background staining.
Although the exact explanation for the beneficial effect of the high-salt concentration
is not known, it may alter the conformation of protein molecules and change their overall
charge, making them less likely to bind nonspecifically on the surface of the section. It is
known that high salt concentrations tend to precipitate proteins out of the solution in bio-
chemical studies and are also used to wash chromatography columns. Accordingly, only
the antibody molecules that have been bound specifically to antigenic sites remain on the
section surface in the presence of high salt concentrations.
If cross reactivity is a problem during conjugated gold-antibody double labeling with
monoclonal antibodies from the same animal (e.g., mouse monoclonals), it can be avoided
by incubating very carefully first one side of the grid in one of the mouse monoclonals and
then the other side of the grid in the second mouse monoclonal (Chiovetti, 1998).
Precaution must be used to prevent sinking of the grid in drops of the incubation reagents.
Hexagonal mesh, uncoated nickel grids should be used.
To avoid the adverse effect of high temperatures on thin resin sections in the
microwave oven, staining can be carried out at ~5°C in the microwave oven (Hernández-
Chavarría and Vargas-Montero, 2001). Heat generated by microwave irradiation is dissi-
pated by this approach. Rapid staining is accomplished by molecular vibrations in the
microwave oven, which induce molecular collisions leading to accelerated chemical reac-
tions. Thin resin sections of the tissue fixed with glutaraldehyde/osmium tetroxide are
transferred onto a grid, which is then placed into a BEEM capsule. Six capsules are placed
on a plastic support, which is placed into a 500-ml beaker containing ice cubes and 300 ml
of tap water, covering the bottom of the capsules. It takes ~5 min to equilibrate the
temperature in the ice bath to 5°C, which is maintained during staining in the microwave
oven. The temperature is measured after each heating period, and ice cubes are added as
melting occurs.
The staining is carried out with of 4% uranyl acetate in 50% ethanol for 1 min
in a microwave oven set at a power level of 125.6W, followed by rinsing with 500 ml of
distilled water. This is followed by staining for 1 min with of triple lead citrate (Sato
et al., 1988) and then rinsing with 500 ml of distilled water. This lead citrate staining solu-
tion avoids the production of artifactual lead carbonate precipitates.
160 Chapter 7
Because epoxy resins copolymerize with tissue proteins, and acrylic resins do not, sec-
tions of the former yield less immunostaining. However, to take advantage of the other
superior characteristics of epoxy resins explained earlier, the immunostaining of sections of
these resins can be enhanced by moderately increasing the proportion of the accelerator
DMP-30 and microwave heating (Brorson, 1998a, b; Brorson et al., 1999). Conventional
concentrations of accelerator in the epoxy mixture form abundant chemical bonds between
resin and tissue. In contrast, a high concentration of accelerator reduces copolymerization
of the epoxy resin with tissue proteins, while heating breaks down both protein crosslink-
ages introduced by aldehydes and the bonds between the resin and the tissue. The break-
down of abundant bonding with heating in the former case is insufficient to allow efficient
access of the antibody to the antigen. Figure 7.3 shows the possible mechanism responsible
for exposing epitopes to antibodies on the surface of thin epoxy sections after heating.
Tissue specimens are fixed overnight at 4°C with a mixture of 4% paraformaldehyde
and 1% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3). They are dehydrated in ethanol
followed by propylene oxide. Infiltration is carried out in two steps using DMP-30 in
concentrations of 4% and 2%, respectively, and embedding in the resin containing 2%
DMP-30. The specimens in gelatin capsules are polymerized for 3 days at 56°C. Thin sec-
tions mounted on nickel grids are treated in 0.01 M citrate buffer (pH 6.0) for 15 min at
95°C in a PCR machine (GeneAmp 2400, Perkin Elmer).
The sections are treated with 10% BSA in PBS (pH 7.2) for 4 hr to block nonspecific
labeling. Incubation is carried out overnight at 4°C in the primary antibody, appropriately
diluted in PBS. This is followed by washing three times for 5 min each in PBS and incu-
bation for at 22°C in colloidal gold (15 nm)–conjugated secondary antibody, appro-
priately diluted in PBS containing 3% BSA. The sections are poststained with 5% uranyl
Antigen Retrieval on Resin Sections 161
acetate in 30% ethanol for 20 min and then with lead citrate for l0 min. The results of this
procedure are shown in Figure 7.4.
EFFECT OF HEATING
0.1% Tween 20. Following rinsing in PBSG containing Tween 20, the sections are rinsed
in PBSG and then in PBS. The sections are postfixed for l0 min with 2% glutaraldehyde
in PBS and rinsed several times in PBS and distilled water; poststaining is carried out with
uranyl acetate and lead citrate. For negative controls, the primary antibody is replaced with
PBS. The results of this procedure are shown in Figure 7.5.
A microwave oven operating at 2,450 MHz with a maximum output power of 900 W
and a cycle time of 2 sec can be used for rapid staining in a microwave oven (Cavusoglu
et al., 1998). The oven contains a gas exhaust system and a built-in ceramic thermocouple
temperature probe (PT 100). To determine the distribution of microwave heating, a piece of
thermal paper is placed on the floor of the oven and subjected to microwave heating at
900 W for 1 min, and the hot spots are located. A glass bottle containing 40 ml of tap water
is placed in the oven to measure the temperature during heating. The temperature of the
water is monitored throughout the staining.
Tissues are fixed with glutaraldehyde followed by and embedded in Epon. Thin
sections are mounted onto a Formvar-coated grid, which is placed (section side down) on
the surface of 4% aqueous solution of uranyl acetate in a staining dish. The dish is placed
on the hot spot in the oven at a power of 600 W for 1 min at 20°C (initial temperature) to
94°C (final temperature). The dish is taken out of the oven and the grid is rinsed with dis-
tilled water. The grid is placed on the surface of lead citrate solution in the dish, which is
placed in the oven and stained for 1 min at 20°C (initial temperature) to 93°C (final tem-
perature). The results of this procedure are shown in Figure 7.6 (Cavusoglu et al., 1998).
Figure 7.7 shows ultrarapid staining of biopsy heart tissue with uranyl acetate and lead
citrate for 15 sec each in a microwave oven.
method requires ~20 hr. All steps are carried out in a microwave oven. Tissues are fixed
in a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol, and embedded in
LR White resin for 75 min. Thin sections are incubated in primary antibody at 37°C for
15 min and then in colloidal gold–goat antirabbit IgG for 15 min at the same temperature
(Rangell and Keller, 2000).
Temperature should be strictly controlled in the microwave oven with a temperature
probe that has a feedback mechanism to regulate the energy output of the microwave oven
and thus maintains the optimal temperature. Alternatively, temperature can be controlled
by placing a water load in the chamber of the microwave oven, which absorbs extra energy
and provides humidity, slowing the evaporation of reagents. In addition, hot spots in the
chamber should be avoided by using the neon bulb display method (Chapter 5).
Labeling in the microwave oven is usually carried out at 37°C for 15 min. Longer
durations and higher temperatures may result in undesirable changes in antibody concen-
tration and molarity of the salts and pH. After heat treatment, the sections should be kept
at room temperature for at least 2 min to stabilize the antibody-antigen complexes. The
step-by-step procedure for microwave heat-assisted immunolabeling of resin-embedded
thin sections for electron microscopy follows (Rangell and Keller, 2000):
Immunogold-Silver Staining
Jackson et al. (1988) were the first to employ the immunogold–silver staining (IGSS)
method in combination with microwave heating. They completed within minutes the
incubations in primary and secondary antibodies for detecting immunoglobulins in paraf-
fin sections of human tonsil. van de Kant et al. (1990) applied the same method, except
that resin instead of paraffin sections were used to detect bromodeoxyuridine incorporated
in cells of the mouse testis. Tissue morphology is preserved better in a resin than in paraffin.
The former also allows the use of thinner sections.
Boon et al. (1989) used a similar procedure for staining beta-human chorionic
gonadotropin in paraffin sections of the syncytiotrophoblast of first-trimester placenta.
Recently, using the IGSS method, Taban and Cathieni (1995) visualized the gold-
protein-ligand complex on cryostat sections of rat brain; this method can be used for light
and electron microscopy.
Droplet Procedure
Tissues are fixed with buffered formalin or Kryofix and embedded in paraffin (Boon
et al., 1991). Sections ( thick) are transferred to a glass slide, deparaffinized, rehy-
drated, and washed in running tap water for l0 min. They are treated with Lugol’s iodine
for 5 min and rinsed briefly in tap water. Following destaining with 2% aqueous sodium
thiosulfate for 10–15 sec, the sections are washed in running tap water for l0 min.
168 Chapter 7
The sections are washed in two changes of 5 min each in Tris buffer I (2.5% NaCl and
0.55% Tween 20, diluted in 0.05 mol/liter Tris-HCl buffer, pH 8.2). The excess buffer is
removed by wiping around the sections, which are then covered for l0 min with of
normal goat serum (NGS) diluted 1:1 with PBS (pH 7.4). Excess NGF is removed by
wiping around the sections.
The sections are covered with of primary antibody appropriately diluted in PBS
(pH 7.4) containing 0.05% BSA (freshly prepared). The slide is placed on the polystyrene
platform in the microwave oven and heated at 50% power for 5 min. A water load of 200 ml
tap water has already been placed in the oven. The sections are washed with Tris buffer for
l0 min, followed by washing in two changes of l0 min each in Tris buffer II (0.05 mol/liter
Tris-HCl buffer, pH 8.2). Excess buffer is removed by wiping around the sections, which
are then covered with of NGS for 10 min at room temperature.
Excess NGS is removed, and the sections are covered with of colloidal gold
conjugated secondary antibody. The slide is placed on the polystyrene platform in the
microwave oven and heated at 50% for 5 min; the oven contains a water load of 200 ml of
tap water. The sections are washed in three changes of 5 min each in Tris buffer II, rinsed
with distilled water, and washed three times of 3 min each in distilled water.
After excess water is removed, the sections are covered with of silver
enhancement mixture for 8–11 min at 20°C. The silver enhancement mixture is prepared
immediately before use by mixing equal volumes of the enhancer and initiator solutions of
the Janssen Intense™ LM kit. The sections are washed three times for 5 min in distilled
water, dehydrated, and mounted.
Chapter 8
169
170 Chapter 8
General Methods of Antigen Retrieval 171
the liquid is at the same temperature as that of the slides. This jar also acts as neu-
tral ballast in the oven, slowing down the speed at which the boiling temperature
is achieved. The buffer is prepared as follows:
Stock solution A: 0.1 M citric acid is prepared by mixing 21.01 g citric acid with
enough distilled water to make 1,000 ml.
Stock solution B: 0.1M sodium citrate is prepared by mixing 29.41 g sodium
citrate with distilled water to make 1,000 ml.
The working solution is prepared just before use by mixing 18 ml of solution A
with 82 ml of solution B plus sufficient distilled water to make 1,000 ml, and
adjusting the pH to 6.0.
The jar containing the slides can be covered with loosely fitting lids or vented
screw caps; do not tightly close the jar or use aluminum foil to cover the jar.
However, covering the jar is not obligatory if it has enough (2–3 cm) empty space
above the buffer level.
9. Place the jars in the center of the oven on a rotary plate to ensure uniform heat-
ing of the slides.
10. Set the power to maximum; a power setting from 7–10 is recommended.
11. Set the time to 10–15 min, and check the buffer temperature with a temperature
probe. The temperature of the buffer is different from that in the oven, so it is dif-
ficult to measure and control the temperature in the jar.
12. When the buffer begins to boil, allow it to cool for 5 min. Count the time of
antigen unmasking from the boiling time. It is necessary to obtain vigorous
boiling. The time for epitope unmasking depends upon the antigen, the antibody,
the tissue type, the type of the fixative, and the duration of fixation. Thus, one
has to standardize microwave heating by trial and error. A known positive
control is essential. As an average, the time for epitope unmasking varies from
2–10 min.
13. Check the level of the buffer in the jar, and restore it between heating cycles.
Compensate the buffer outflow with the buffer, while replenishing evaporated
buffer with distilled water. The slides must remain fully immersed in the buffer.
14. After 5 min, again set the time to 5 min and restart the oven.
15. Repeat steps 12 and 13.
16. Remove the jar from the oven, and allow it to cool at room temperature for
20 min in a fume hood.
17. Rinse several times in 0.05 M PBS (pH 7.5).
18. Discard the used buffer.
19. When the DAB method is used, inhibit endogenous peroxidase by treating the
sections with of 30% in 50 ml of PBS for 30 min, followed by thor-
ough washing in PBS.
20. Treat the sections with a mixture of 3% normal serum and 0.4% Triton X-100 for
~30–60 min at room temperature to aid antibody penetration and block back-
ground staining.
21. Drain the excess serum from the slides, and incubate overnight at 4°C in a humid
chamber with the primary antibody diluted appropriately in PBS. Incubation
should be carried out with stirring to promote antigen-antibody contact.
22. Rinse in three changes of PBS.
172 Chapter 8
23. Incubate for 30 min at room temperature in the linking agent (biotinylated anti-
immunoglobulin; Vector Laboratories, Burlingame, CA).
24. Rinse in three changes of PBS.
25. Incubate for 45–60 min in avidin-biotin peroxidase or alkaline phosphatase or
ABC Elite or a third antibody if using the double indirect method.
26. Rinse in three changes of PBS.
27. Develop the color in 0.02–0.05% DAB activated with 0.003–0.01% in 0.1 M
Tris buffer for 5–15 min. This step must be carried out in a fumehood. If using
the alkaline phosphatase method, use the Alkaline Phosphatase Developing kit
(Vector Red).
28. Wash in running tap water for 10 min.
29. Counterstain lightly for 20 sec with hematoxylin, and rinse in water.
30. Dehydrate in ethanol, clear in xylene, and cover-slip with Permount.
Note: Instead of coating the slides with an adhesive, use SuperFrost Plus slides (Fisher
Scientific). These slides are charged positively and the sections are charged negatively,
thus, preventing the sections from detaching from the slides while boiling. If these slides
are unavailable, ordinary glass slides can be coated with an adhesive such as poly-L-lysine
(0.1%) or neoprene (Aldrich Chemical, Milwaukee, WI). However, the use of any type of
adhesive on the slide may not prevent detachment of sections from the slide. The main rea-
son for losing sections during boiling is the presence of air bubbles between the section
and the slide, not the lack of adherence of the section to the slide.
To avoid the risk of drying the sections during microwave heating, it is necessary to
heat them in multiple 4- to 5-min cycles and to replenish the jars between heating periods.
Some evidence indicates that drying of sections on glass slides prior to histological stain-
ing in a microwave oven instead of in a conventional oven or on a hot plate, has several
advantages: paraffin sections adhere better to the glue-coated slides, drying time is reduced
from 1 hr to 1 min, and nonspecific background staining may be reduced.
It has been suggested that drying of paraffin sections first at 38°C and then at higher
temperatures improves immunostaining of the proliferating cell nuclear antigen (Golick
and Rice, 1992). Additional studies are required to evaluate the relationship between the
temperature of slide drying and the extent of immunostaining. Another suggestion is that
mild boiling of the epitope retrieval fluid gently affects tissue sections, so they are less
likely to be dislodged from the slide. The microwave oven should be left at high rather than
changed to a medium setting because a change of setting does not affect the wavelength or
actual power of the microwaves generated.
It is thought that the intensity of specific immunostaining can be enhanced and back-
ground staining simultaneously reduced by gentle orbital rotation (using a serological rota-
tion) of slides during manual incubations (Butz et al., 1994). Another advantage of this
approach is shortening antibody incubation times without sacrificing sensitivity.
Extreme antigen enhancement may cause false-positive staining. Such staining has
been observed in the case of p53 antigen (using monoclonal antibody D07) with Target
Unmasking Fluid (TUF) containing 35% urea in the microwave oven at 96°C for 30 min
(Baas et al., 1996). This and other evidence indicates that there is a limit to the extent to
which antigen enhancement can be applied to achieve optimal detection of a given antigen.
If necessary, the slides with sections of paraffin-embedded tissues can be reused to
detect a second type of antigen when the staining of the first type of antigen is negative.
General Methods of Antigen Retrieval 173
This need may arise when a limited number of slides is available. For this type of study,
heat-induced antigen retrieval should be carried out only once. One such treatment lasts
for many months. Repeated use of antigen retrieval tends to cause background staining.
Such an approach has been successfully used for staining epithelial membrane antigens
and cytokeratin in the tonsil tissue fixed with Bouin’s fixative (Roche et al., 2000).
results for keratin and p53 antigens in 3-mm-thick paraffin sections of archival tissues
(Arnold et al., 1996). Acid formalin is less effective as a protein crosslinking agent, as it
protonates amino groups and thus allows easy access of the antibodies to the epitopes. In
this study, buffered formalin produced the least satisfactory results, which was expected
because the fixative forms protein crosslinks more rapidly and effectively. Nevertheless,
note that the above-mentioned fixatives, other than buffered formalin, produce compara-
tively poor morphological details.
Procedure
citrate buffer (pH 6.0) and heated for l0 min at 100°C on a hot plate (PC-351) (Corning,
Utica, NY). The slides are allowed to cool at room temperature for 20 min. After being
washed in PBS, the sections are incubated overnight at 4°C in the monoclonal antibody
clone E12 (DAKO, Carpinteria, CA) and diluted 1:4000 with PBS. They are washed
with PBS and then treated with the Elite ABC system according to the directions of the
manufacturer (Vector, Burlingame, CA). The chromogen is developed with 3-amino-9
ethyl carbazole (Sigma) at room temperature. After being washed with PBS, the sections
are counterstained with Mayer’s hematoxylin and mounted in Glycergel (Sigma). The
results of this procedure are shown in Figure 8.3.
Conventional light microscopy of multifocal lesions of VIN may not show VIN 3 and
instead may show VIN 2, VIN 1, or even normal squamous epithelium (van Beurden et al.,
1998). Interobserver variation in the interpretation of the grading of VIN is not uncommon.
To determine correct treatment of the patient, it is necessary to know which lesions show
VIN 3 and which do not. The standard treatment for VIN 3 is drastic surgical excision of
all visible lesions. However, an alternate approach is taking multiple biopsies; the removal
of involved skin using cold knife surgery or laser vaporization without radical surgery (van
Beurden et al., 1998).
Interobserver variation in the grading of VIN has been observed when using
hematoxylin-eosin only, MIB-1 monoclonal antibody alone, or combined hematoxylin-
eosin and MIB-1 antibody (van Beurden et al., 1999). This antibody is the most versatile
proliferation worker for the sections of formalin-fixed and paraffin-embedded tissues.
Normal vulvar skin and VIN lesions are fixed with formalin and embedded in paraffin
according to standard procedures.
Following deparaffinization with xylene and rehydration with ethanol, the slides are
placed in 0.01 M sodium citrate buffer (pH 6.0) and boiled for l0 min on a hot plate; after
cooling for 20 min to room temperature the slides are subjected to three different treatments:
1. Staining with hematoxylin-eosin
2. Incubation in MIB-1 antibody
3. Incubation with a combination of MIB-1 antibody and hematoxylin-eosin
Uncertainty regarding the grading of VIN is significantly decreased when MIB-1
antibody is used.
are recommended for immunostaining on thick and thin sections using light and electron
microscopy, respectively, and also for cell smears.
Human brain tissues, for example, are fixed postmortem with 10% formalin for 24–48 hr,
dehydrated, and embedded in paraffin. Sections, cut with a rotary microtome, are mounted
on coated glass slides. The sections are rinsed three times for 5 min each with 0.1 M
sodium phosphate buffer (pH 7.4) and then transferred to 10–15 mM sodium citrate
General Methods of Antigen Retrieval 179
(pH 8.5–9.0; preheated to 80°C in a water bath). They are cooled to room temperature, fol-
lowed by rinsing three times for 5 min each in the buffer. The sections are immersed in
0.3% nonfat dry milk in buffer containing 0.3% Triton X-100 and 0.01% sodium azide for
30–60 min.
The sections are incubated in a primary antibody (diluted appropriately) for 72 hr at
4°C in a sealed humid chamber; the incubation is carried out by applying droplets of the
antibody to the sections. After being rinsed in the buffer, the sections are incubated for
90 min in secondary antiserum diluted 1:50 with PBX (0.3% Triton X-100, 0.01% sodium
azide and 0.1 M phosphate buffer) and then treated for 1 hr under agitation in peroxidase-
antiperoxidase (PAP), diluted 1:100 with PBX, in a sealed humid chamber in both cases.
The sections are rinsed sequentially with the buffer and then distilled water. They are
incubated for 10 min with continuous agitation in 50ml of 0.05 M imidazole/0.05 M
cacodylate buffer (pH 7.2) containing 50 mg of DAB, followed by an additional 10-min
incubation after adding of 3% hydrogen peroxide with continuous agitation. After
being washed in distilled water, the sections are placed in the buffer, dried, dehydrated,
and cover-slipped with Permount. Note that the ABC procedure can also be used for
immunolabeling.
For assessing the antigen retrieval effectiveness, the staining intensities observed
under the microscope can be divided into five grades: + + + +,+ + +,+ +,+ or – for
180 Chapter 8
very intense, intense, moderate, weak, or negative, respectively. These judgments are made
qualitatively by comparing one section to another.
Loss of methyl groups in DNA is not uncommon in human carcinomas such as colon
adenomas and adenocarcinomas. A strong correlation is found between the malignant phe-
notype and DNA methylation. It is known that 5-methylcytidine (a spontaneous frequent site
of C and T mutation) is involved in the control of gene expression in carcinogenesis and in
tumor progression. Consequently, global DNA hypomethylation could induce protoonco-
gene expression, whereas hypermethylation could silence tumor suppressor gene (Little and
Wainwright, 1995). Monoclonal antibodies can be used to recognize the presence of
a methyl group on the C of cytidine to investigate DNA methylation in situ. An immuno-
histochemical method has been reported for correlating the histopathological pattern with
the immunostaining intensity of the nuclei (Hernández-Blazquez et al., 2000).
Qualitative and quantitative differences can be observed and measured between the
normal and malignant part of each tumor specimen. Morphologically altered nuclei dis-
play densely labeled spots within faintly labeled areas, whereas normal nuclei are darker
and uniformly stained.
General Methods of Antigen Retrieval 181
Procedure
Malignant lesions and normal tissue biopsies (colon) are fixed with formalin and
embedded in paraffin (Hernández-Blazquez et al., 2000). Paraffin sections thick)
are deparaffinized, rehydrated, and rinsed in PBS. They are placed in 0.1 mM citrate buffer
(pH 3.4), heated for 10 min in a microwave oven at full power (720 W), and washed
in PBS. The slides are immersed in 2N HC1 for 2 hr at 37°C and then rinsed in PBS.
The sections are covered with of hybridoma supernatant containing anti-5-MeCyd
monoclonal antibody and incubated for 1 hr at room temperature with a
biotinylated goat antimouse secondary antibody diluted 1:200 in PBS containing
0.1% BSA.
The sections are rinsed in PBS and then treated for 30 min with streptavidin-peroxidase,
diluted 1:100 with PBS-BSA. They are rinsed in PBS and treated with for
5 min. The PBS used for rinsing contains 0.1% Tween 20.
Procedure
Surgically removed tissues are fixed with 4% buffered formalin and embedded in
paraffin (Turner et al., 1999). Sections thick) on slides are deparaffinized, rehy-
drated, and then treated with 3% to block endogenous peroxidase. They are placed in
0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven. An EPOS rabbit anti-
human Ki-67 antibody is applied as supplied (DAKO); it is ready to be used without any
dilution. Color development is accomplished with metal-enhanced DAB for 15 min, fol-
lowed by light counterstaining with hematoxylin. Quantification of Ki-67 antibody-labeled
182 Chapter 8
cells is performed with an image analysis system (VIDAS 21), and the tumor cell nuclear
staining is recorded as the percentage of positive cells (labeling index).
The following method was used for double immunostaining of fast and slow skeletal
muscle fibers in the same section (Carson et al., 1998). Muscle specimens (2–4 mm) are
fixed in 10% neutral buffered formalin for 3 hr and then immersed in 0.1 M PBS contain-
ing 17% sucrose for 3 hr. They are embedded in paraffin, and 4 to sections are
mounted onto Superfrost Plus slides. The sections are deparaffinized and placed in Target
Unmasking Fluid (TUF) preheated in a microwave oven and maintained at 90°C for
15 min (without boiling) and slowly cooled to room temperature. The sections are rinsed
three times for 3 min each in PBS and then placed in 3% hydrogen peroxide in PBS for
30 min to block endogenous peroxidase, followed by again rinsing three times in PBS.
The sections are blocked with 10% nonimmune goat serum for 30 min at room tem-
perature and then rinsed in PBS. They are incubated overnight at 4°C in monclonal anti-
body against MHC-I (diluted 1:20 in PBS) in a humid chamber, followed by rinsing three
times in PBS. The sections are incubated for 1 hr in the goat antimouse biotinylated
secondary antibody in a humid chamber.
The sections are treated for 1 hr with streptavidin-alkaline phosphatase in a humid
chamber and then washed in PBS. They are developed for 10 min in nitroblue tetra-
zolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP). After washing in PBS, the
sections are treated for 30 min in a double-staining enhancer (Zymed Laboratories, San
Francisco, CA) to prevent the first stain from reacting with the second. They are thor-
oughly rinsed in distilled water followed by PBS.
The sections are treated for 30 min with 10% nonimmune goat serum and then rinsed
in PBS. This is followed by overnight incubation at 4°C in monoclonal antibody against
General Methods of Antigen Retrieval 183
MHC-II (diluted 1:10 in PBS) in a humid chamber and rinsing in PBS. The sections are
incubated for 1 hr in the goat antimouse biotinylated secondary antibody in a humid cham-
ber. They are treated for 1 hr with streptavidin–alkaline phosphatase in a humid chamber
and then washed in PBS. The sections are treated with 3-amino-9-ethyl-carbazole (AEC)
chromogen; the staining intensity is monitored microscopically. The staining duration
is ~10 min. They are counterstained for 10 min in NBT/BCIP, washed in distilled water;
and cover-slipped with an aqueous mounting medium. An immunostaining kit (Histostain-
SP) is commercially available (Zymed Laboratories, San Francisco, CA).
Procedure
Cyclin D1 (PRAD-1, bcl-1) protein plays an important role in the transition of cells
from resting phase to DNA replication phase. This protein has oncogenic properties, and
its overexpression is thought to play a role in tumorigenesis. Overexpression of cyclin D1
mRNA and protein has been demonstrated in a variety of lymphoid as well as nonlym-
phoid neoplasms (de Boer et al., 1995; Vasef et al., 1997a, b; Naitoh et al., 1995). The
overexpression of cyclin D1 protein has also been demonstrated in neoplastic proliferating
parathyroid tissue, adenomas, and nonneoplastic proliferating parathyroid gland, but sel-
dom in normal parathyroid tissue (Vasef et al., 1999). It is suggested that PRAD-1 gene
alteration is responsible for cyclin D1 protein overexpression in parathyroid hyperplasia.
The mechanism underlying such gene alterations remains undefined. Although cyclin
D1 is not useful in distinguishing parathyroid carcinomas from parathyroid adenomas,
this protein is useful in distinguishing between hyperplasia and normal parathyroid
glands in histologically ambiguous cases. The following immunohistochemical method is
recommended for the localization of cyclin Dl in neoplastic parathyroid tissue (Vasef
et al., 1999).
Biopsy tissue specimens are fixed with formalin and embedded in paraffin. Sections
thick) are mounted on silanated slides, heated at 56°C for 1 hr, deparaffinized in
xylene, rehydrated in graded ethanols, and rinsed in distilled water. They are placed in 0.01
M citrate buffer (pH 6.0) and heated in a microwave oven for six cycles of 5 min each, fol-
lowed by cooling at room temperature for 20 min. Endogenous peroxidase activity is
blocked with hydrogen peroxide in distilled water for 8 min, and nonspecific background
staining is prevented by treatment with nonimmune horse serum for 20 min.
General Methods of Antigen Retrieval 185
Procedure
Procedure
Tissues are fixed in formalin, embedded in paraffin, and sections thick) are trans-
ferred onto Superfrost Plus–coated slides (Mason et al., 2000). The sections are deparaf-
finized with xylene and then rehydrated with descending concentrations of ethanol. They
are placed in 0.1 M sodium citrate buffer (pH 6.0) and heated in a microwave oven at full
General Methods of Antigen Retrieval 187
power. The sections are rinsed in PBS and then incubated with a mixture of two primary
antibodies for ~30min at room temperature, using appropriate dilutions that have been
determined by titration. The pair of primary antibodies are either from different species
or of differing Ig isotypes/subclasses. They are washed in TBS and incubated for 1 hr in
the dark in secondary antibodies, one conjugated to fluorescein isothiocyanate (FITC) and
the other to Texas Red. This is followed by washing in TBS and then counterstaining for
30 sec with DAPI (1 mg/ml of an aqueous solution diluted 1:100 in absolute ethanol). The
sections are rinsed in tap water and mounted in antifading medium (DAKO). The slides
should be kept at 4°C in the dark when not viewed immediately.
Procedure
(diluted in 0.05 M Tris buffer [pH 7.4] containing 0.15M NaCl: TBS) for 30 min
at room temperature.
4. Antisera or monoclonal antibodies are applied by diluting as required in TBS
containing 0.25% BSA (TBS-BSA). Monoclonal antibodies are applied for 1 hr
at room temperature, while polyclonal antibodies are applied overnight at 4°C.
5. The sections are rinsed three times for 5 min each in TBS containing 1% Triton
X-100. The addition of this detergent is optional but may sometimes reduce back-
ground staining.
6. Fluorescence-labeled second antibody is applied for 30 min at room temperature
by diluting in TBS-BSA as required. Fluorscein isothiocyanate-(FITC)–Texas
Red– or aminomethyl coumarin (AMCA)–labeled variants can be used.
7. The sections are rinsed three times for 5 min each in TBS with or without 1%
Triton X-100. If this detergent is used, the sections are rinsed three times for 5
min each in TBS without the detergent before microwaving.
8. Place five slides with sections in a plastic jar containing 50 ml of 10 mM sodium
citrate buffer (pH 6.0). The jar is placed in a tray containing 1 liter tap water and
microwaved at 780 W. Three cycles of microwaving for 5 min each are adequate.
More cycles may lead to losses of antibodies from the sections and fewer cycles
may cause inefficient blocking of antibody cross-reactivity. The optimal condi-
tions of microwaving differ depending on the antibody-antigen under study.
9. After microwaving, the slides are left in the citrate buffer at room temperature for
20 min.
10. The sections are rinsed in TBS and then incubated in the primary antibody, which
may be raised in the same species as those used in step 1 above.
11. This is followed by rinsing three times for 5 min each in TBS with or without 1%
Triton X-100 (cf. step 5).
12. A new round of second antibodies labeled with a fluorophore other than that in
step 6 is applied. Alternatively, a triple-layer method employing a second layer
of biotin-labeled antiimmunoglobulins followed by fluorescence-labeled strepta-
vidin can be used.
13. They are rinsed three times for 5 min each in TBS with or without 1% Triton
X-100. If the detergent is used, at least the final rinse should be in TBS without
the detergent.
14. The sections are mounted in a suitable antifade medium such as VectaShield
(Vector Laboratories, Burlingame, CA).
15. They are observed with a fluorescence microscope equipped with selective filters
for the fluorophores used. An example of the double staining is shown in Figure 8.7
(Plate 4B, C, D).
Control Procedures
fluorescence characteristic of the label introduced in step 12. If they do, microwaving is
insufficient and has not completely blocked the free antigen combining sites presented on
the second antibodies introduced in step 6. These sites, if not blocked by microwaving, will
bind to immunoglobulins present in the normal serum. These immunoglobulins in turn will
bind the second round of second antibodies. Thus, all sites marked by the first antibody
will fluoresce with a mixed color characteristic of the two fluorophores used in steps 6 and
12, respectively. Alternatively, it is possible that the first round of second antibodies may
not have saturated all binding sites on the primary antibodies, leaving these free to bind
second antibodies added in the second staining cycle. In any case, successful microwaving
blocks cross-reactivity.
Immunoenzymatic Detection
mantle cell lymphoma (Brynes et al., 1997). Compared with HIER or SIER method alone,
the combined approach produced stronger immunostaining and lower background stain-
ing. Sections of formalin-fixed tissues are mounted on glass slides and heated in a
slide oven for 1 hr at 56°C. They are deparaffinized with xylene and rehydrated with
ethanol to distilled water.
The slides are placed in a plastic microwavable rack and immersed in a microwavable
staining dish filled with 200 ml of 0.01 M citrate buffer (pH 6.0). Blank slides are added to
the rack to maintain a uniform volume in the container. The container is covered at an
angle with the lid and heated twice for 5 min per cycle at 800 W in a microwave oven.
Distilled water (50ml) is added after the first heating cycle to cool at room at room tem-
perature for 10 min.
The buffer (~800 ml) is heated to boiling in the microwave oven and poured into an
80-W ultrasonic cleaner (Bransonic 12, Branson Cleaning Equipment Co., Shelton, CT).
The slide rack is sonicated for 1 min and then cooled in buffer in the staining container for
an additional 10 min.
The sections are treated with Biotek enzyme (Ventana Biotek) for 10 min, followed
by blocking of endogenous peroxidase with 3% hydrogen peroxide in buffer for ~8 min.
Immunostaining is carried out in an automated immunostainer (TechMate 1000 Equipment
Co., Shelton, CT). The slide rack is sonicated for 1 min and then cooled in buffer in the
staining container for an additional 10 min.
A cocktail (1:1) of two monoclonal anticyclin D1/bc l-1 antibodies were used:
P2D11F11 (diluted 1:40) and 5D4 (diluted 1:100); these two antibodies can be obtained from
Vecta Laboratories, Inc., Burlingame, CA, and Immunotech, Westbrook, ME, respectively.
The avidin-biotin immunoperoxidase detection system employing DAB is used as the chro-
mogen (Ventana Biotek). After counterstaining in dilute Mayer’s hematoxylin, the sections
are dehydrated and mounted in Permount.
In certain cases maximally effective antigen retrieval conditions tend to cause non-
specific staining and/or background staining. This artifact can be avoided by employing a
combination of mild heating and enzyme digestion. This approach can also be used for
multiple immunostaining, including that of the PCNA (Ezaki, 2000).
Tissue specimens are fixed with 4% paraformaldehyde and embedded in paraffin at
60°C for 1 hr. Sections thick) are mounted on gelatin-coated glass slides, deparaf-
finized, and rehydrated in distilled water. They are treated with 0.005% pepsin for 15 min
at 37°C, followed by heating in 0.01 M citrate buffer (pH 6.0) in a microwave oven (300
W) at 80°C for 15 min. The sections are washed in distilled water for 5 min, rinsed in
0.01 M PBS (pH 7.2) for 15 min, and treated with 0.3–1% to quench endogenous
peroxidase activity.
They are incubated in the primary antibody (PC10, diluted 1:200 in PBS containing
0.2% BSA for PCNA) for 1–2 hr at room temperature. The sections are washed five times
for 5 min each with PBS and then incubated in the enzyme-conjugated secondary antibody
in PBS containing 1% heat-inactivated normal rat serum for 1 hr. Horseradish peroxidase
General Methods of Antigen Retrieval 191
2-MERCAPTOETHANOL–SODIUMIODOACETATE–ASSISTED
ANTIGEN RETRIEVAL
primary antibody with the antigen and substitution of the primary antibody with non-
immune serum from the same species.
Endogenous peroxidase activity is quenched by treatment with 0.5% in methanol,
immunostaining is carried out using a Vectastain Elite ABC kit, and visualization of the
secondary antibody is achieved with DAB enhanced with nickel (Vector Laboratories).
Procedure
the bovine prion protein. The epitope has been mapped to the sequence IHFG. This epi-
tope is also conserved in the human prion protein sequence located at AA 139–142. This
antibody shows stronger sensitivity and specificity than those obtained with 3F4 antibody
for the human prion protein (Van Everbroeck et al., 1999). The antibody F89/160.1.5 is
used at a dilution of 1:20,000 with TBSB (150 mM NaCl, 1% BSA, 50 mM Tris-HCl
buffer; pH 7.4) (final concentration is 55 ng/ml). The antibody 3F4 is used at a dilution of
1:2,000 with TBSB (final concentration is 1:25 mg/ml).
Procedure
Brain tissue specimens are treated with 98% formic acid for 1 hr to reduce infectivity
and embedded in paraffin. Sections thick) are picked up on 0.1% poly-L-lysine-coated
glass slides; Superfrost Plus slides are not recommended because sections tend to be dis-
lodged during multiple treatments. The sections on slides are deparaffinized with xylene and
rehydrated with descending concentrations of ethanol. They are treated with 5% picric acid
for 15 min at room temperature and then thoroughly washed with tap water. The sections are
exposed to 0.3% in methanol for 30 min to block endogenous peroxidase. This is fol-
lowed by autoclaving for 10 min at 121°C using 10 mM citric acid (pH 6.0) as the recovery
buffer. The sections are allowed to cool and then washed in distilled water. The sections are
treated with 88% formic acid for 5 min and then washed in distilled water. Finally, precooled
(4°C) guanidine thiocyanate (4M) is pipetted onto the sections and incubated for 2 hr at 4°C.
The sections are exposed to normal swine serum (1:25) in TBSB (150 mM NaCl, 1%
BSA, 50 mM Tris-HCl; pH 7.4) for 30 min to block nonspecific binding sites. They are
incubated overnight in a humid chamber at room temperature in the primary monclonal
antibody (3F4) (Senetk, St. Louis, MO) and diluted 1:2000 with the buffer. The avidin-
biotin complex (ABC) method is used to detect antibody binding. The bound antibody is
detected by incubation with the secondary antibody (biotinylated goat antimouse IgG
diluted 1:100 in TBSB) for 30 min at room temperature. This is followed by incubation for
1 hr with avidin-biotin-horseradish peroxidase complex, diluted 1:200 in TBSB. As the
staining mixture, 0.05% DAB in TBS (150 mM NaCl, 50 mM Tris-HCl; pH 7.6) with
0.002% is used for 5 min. After thorough washing for 10 min in running tap water,
the sections are counterstained with Harris’ hematoxylin for 30 sec. They are dehydrated
and mounted. The results of this procedure are shown in Figure 8.9.
Great care should be taken in handling the tissue to avoid infection. Picric acid is both
toxic and explosive. Safety guidelines must be used when working with this reagent.
Guanidine thiocyanate is also a biohazardous material.
employing these two reagents sequentially, two nonoverlapping antigens can be localized,
one exhibiting brown color and the other black.
Simultaneous detection of three antigens within one tissue section became possible
by employing an additional peroxidase substrate such as the Vector VIP Substrate kit
(Vector Lab, Burlingham, CA) (Pujic et al., 1998). This substrate is oxidized by horseradish
peroxidase and yields a rose-colored final reaction product which differs in color from that
196 Chapter 8
Procedure
picogram quantities of specific antigens (Zijlstra and Schelling, 1999). Two conventional
methods for detecting multiple antigens are (1) individual antibodies against different antigens
used for separate cell structures and (2) multiple primary antibodies applied against different
antigens in the same cell culture and visualized with separate staining methods. Zijlstra and
Schelling (1999) have developed the following simple procedure for detecting and quantify-
ing four individual fibronectin isoforms within a single fibroblast monolayer culture.
Procedure
Cells are cultured in 100-mm (internal diameter) tissue culture dishes, rinsed three
times at 37°C with PBS, and fixed with 100% methanol for 20 min at –20°C. The
methanol is vacuum-aspirated, and residual methanol is allowed to evaporate. A grid
(55 X 60 mm) is formed by placing the culture dish on top of a template and tracing the
lines using a PAP-pen (the Binding Site, San Diego, CA). Thus, the surface of the dish is
divided into 20 distinct areas (11 X 15 mm). The spacing of the area in these grids allows
the use of a multichannel pipette. The PAP-pen lines provide a hydrophobic barrier
between each area and prevent horizontal movement of fluids.
The culture is incubated in methanol containing 3% for 10 min to quench
endogenous peroxidase activity. The cells are rehydrated in 70% ethanol for 2 min, fol-
lowed by rinsing for 5 min in PBS. Nonspecific binding is blocked by incubating the cells
for 1 hr in the blocking buffer (1% BSA in PBS).
The four monoclonal antibodies used and their specificity are shown in Table 8.1.
Approximately of the primary antibodies, appropriately diluted in blocking buffer,
are applied to individual areas of the monolayer. The culture dish is placed inside a humid
chamber and incubated for 2 hr at room temperature. The template of the grid is used to
determine the location, quantity, and dilution of each antibody. Each area is rinsed with
of PBS, followed by three changes for 5 min each with blocking buffer A
HRP-conjugated secondary antibody, diluted in blocking buffer, is applied to each area for
1 hr in a humid chamber. The areas are rinsed with of blocking buffer. Approximately
of DAB is applied for 5 min, rinsed twice with distilled water, and counterstained
with Erlich’s hematoxylin. All washing and blocking solutions are applied with a multi-
channel pipette and removed by aspiration using a 1-ml syringe attached to a vacuum trap.
The immunostaining is documented with a Sony DKC-5000 digital photosystem. In the
above method, antigen retrieval is not required.
198 Chapter 8
The use of freshly frozen brain tissues in immunohistochemical studies has certain
drawbacks, such as poor preservation of tissue morphology and antigenicity resulting from
ice crystal formation. Many antibodies available for immunohistochemistry have not been
applied to this type of tissue. Microwave heating at boiling temperature is effective in the
rapid retrieval and immunostaining of antigens in freshly frozen tissues, including neural
tissues. This technique has been used to enhance staining of the glial fibrillary acidic pro-
tein (GFAP) in rat brain tissue using monoclonal anti-GFAP antibody (DeHart et al.,
1996). Uniform increased staining of cell bodies and large astrocytic processes occurs in
General Methods of Antigen Retrieval 199
both grey and white matter without excessive background staining. If nonuniform staining
is observed, it may be due to uneven section thickness or unevenly frozen tissues.
Procedure
Fresh rat brain tissue is immediately immersed in 2-methyl butane at –15 to –25°C
for several minutes and stored at –70°C. Sections are cut in the parasagittal plane
on a cryostat, which are thaw-mounted onto poly-L-lysine-coated glass slides and stored at
–70°C. The sections are dried on a slide warmer at the lowest setting to avoid excessive
drying. They are fixed with 4% formaldehyde in PBS (pH 7.5) for 30 min and then rinsed
three times for 10 min each in PBS to remove excess fixative. This is followed by treatment
with 0.3% hydrogen peroxide in PBS for 30–40 min to quench endogenous peroxidase
activity.
After being washed in three changes of 5 min each in PBS, the slide is placed in a
plastic jar containing 60ml of 10 mM sodium citrate buffer (pH 6.0) and 0.04% Triton
X-100. The jar is loosely covered with its screw cap and heated for 5 min in a microwave
oven at high power. The buffer starts to boil after ~90 sec. The heating process is inter-
rupted at intervals of 1 min so that the fluid level can be checked and replenished in the jar.
The sections must not be allowed to dry. The jar is removed from the oven and allowed to
cool to room temperature for 20–30 min. The sections are rinsed in three changes of 5 min
each in PBS.
The sections are treated with 10% normal horse serum for 3 hr at 4°C to reduce non-
specific binding. After being rinsed three times for 5 min each in PBS, the sections are
incubated in the monoclonal anti-GFAP antibody and diluted 1:400 in PBS for 18 hr at
4°C. Following washing three times for 10 min each in PBS, the sections are incubated in
biotinylated secondary antibody diluted 1:200 in PBS for 1 hr at room temperature. The
peroxidase bridge is completed by treating the sections with an avidin-biotin peroxidase
complex solution for 30 min at room temperature. The sections are rinsed twice in PBS,
and immunoreactivity is visualized using DAB (10 mg/15 ml) and 0.024% hydrogen
peroxide in 50 mM Tris buffer, pH 7.6). After a rinse in distilled water, the sections are
dehydrated and cover-slipped.
Procedure
It is known that antibody labeling efficiency over tightly packed antigens is reduced
because of steric hindrance.
Procedure
Small pieces of tissue are fixed for 1 hr in a mixture of 4% formaldehyde and 0.5%
glutaraldehyde in 100 mM phosphate buffer (pH 7.4) (Chicoine and Webster, 1998). They
are infiltrated with 2.3 M sucrose for 24 hr at 4°C, mounted with specimen pins (Leica,
Deerfield, IL), and frozen by immersion in liquid nitrogen. The frozen tissue is sectioned
at –110°C using an Ultracut E ultramicrotome (Leica) equipped with a diamond knife and
an FC4 cryoattachment. Cryosections ~60–80 nm thick are thawed and then mounted on
Formvar-carbon-coated grids.
A microwave oven equipped with Pello 3420 Load Cooler attachment is set at 25°C
and full power. Two beakers, each filled with 500 ml of water, are placed in previously
determined hot spots in the microwave oven (see pages 102–103). Grids containing the
thin cryosections are floated (section side down) sequentially on small drops of 0.15%
glycine and 1% BSA for 15 sec and 5 min, respectively. The reagent drops are placed on a
clean disposable plastic surface which has been placed on the cold spot in the oven (an area
between the two beakers of water). The local temperature is controlled using the
microwave temperature probe immersed in a tube of water placed close to the grids.
The sections are floated on small drops of appropriately diluted primary antibody and
intermittently microwaved for a total duration of 8 min: 2 min with microwave oven on,
2 min with microwave oven off, 2 min with oven on, and 2 min with oven off. This is fol-
lowed by washing twice with PBS for 15 sec in a microwave oven. The sections are treated
outside the oven for 15 min with protein A–colloidal gold (10 nm) complex diluted 1:20 to
1:50 (determined by spectrophotometry) in PBS containing 1% BSA. They are washed in
PBS followed by distilled water and counterstained outside the oven.
Alternatively, the sections are microwaved for 6 min before incubation with the pri-
mary antibody. The remaining steps are also carried out outside the microwave oven.
To detect apoptosis in paraffin tissue sections, the TUNEL technique is most com-
monly used. However, this technique is not specific because it also detects nonspecific
DNA degradation in autolysis or necrosis, and DNA breaks during DNA repair, resulting
in false-positive staining (Suurmeijer et al., 1999). Alternatively, the apoptotic pheno-
type can be visualized by immunostaining target proteins cleaved by caspases, because
the process of apoptosis is irreversible once the caspase cascade is completely
activated. Immunodetection of caspase-cleaved cytokeratin 18 has been accomplished
(Caulin et al., 1997). However, this protocol is specific for detecting cytokeratin
18 in apoptotic cells in epithelial tissues or tumors, although immunostaining of cleaved
actin filaments is more useful because of its omnipresence in human apoptotic cells.
However, whether actin cleavage by caspases is a universal mechanism in apoptosis has
not been established as yet.
202 Chapter 8
IMMUNOHISTOCHEMICAL LOCALIZATION OF
PROSTATE-SPECIFIC ANTIGEN
is no reason to use the terms Skene’s gland and/or paraurethral ducts and glands for the
human female prostate 1999, and Ablin, 2000).
Immunohistochemical studies demonstrate that PSA is expressed in the highly special-
ized, apically superficial layer of male and female secretory cells of the prostate gland, as well
as in uroepithelial cells at other sites of the urogenital tract of both sexes. Such studies pro-
vide evidence that PSA plays a crucial role in the identification of normal or pathologically
altered prostate tissue. In clinical practice, PSA is a valuable marker for diagnosing and mon-
itoring prostate cancer in both sexes. and Ablin (2000) have reviewed the functional-
morphological and some clinical aspects of the normal and pathological female prostate.
Prostate-specific antigen is also a serum marker for prostate cancer. The serum PSA
is generally proportional to tumor volume and correlates positively with the clinical stage
of the disease. Progression of prostate cancer to androgen independence is commonly
associated with a rebound of serum PSA. Prostate-specific antigen elevation in hormone-
refractory prostate tumors is attributed to mutations and/or amplifications of AR, which
broaden its ligand specificity and/or enhance tumor cell’s responsiveness to androgen,
respectively. Hormone-refractory prostate cancer is one of the most detrimental diseases
affecting men in the United States. In males a range of 1–2 ng/ml in serum is normal in the
majority of cases, while values above 3–4 ng/ml are indicative of prostate cancer, benign
prostate hyperplasia, or prostatitis. It is noted, however, that exceptions do occur and that
up to 40% of individuals with levels <4 ng/ml may have prostate cancer.
A healthy female with a normal prostate is characterized by a broad range of serum
PSA values from practically unappreciable amounts to the highest reported ones of
0.9 ng/ml. This value is very close to the normal reference range in the male and
Ablin, 2000).
IMMUNOHISTOCHEMISTRY
Procedure
CARBOHYDRATE ANTIGENS
Molecules other than proteins are also antigenic to various degrees. However, proteins
are the most effective antigens because of their size and structural complexity, and almost
all proteins larger than 1kDa are antigenic. Complex carbohydrates, especially those
bound to proteins or lipids (e.g., glycoproteins and glycolipids) are also of immunological
importance. Simple polysaccharides, such as starch and glycogen, are usually not effective
antigens because they are rapidly degraded within cells and, in addition, do not form
structurally stable epitopes.
Lipids are poor antigens because of their wide distribution, relative simplicity, structural
instability, and rapid metabolism. However, when linked to proteins or polysaccharides they
may function as haptens. Nucleic acids are also poor antigens because of their relative sim-
plicity and flexibility and rapid degradation. Nevertheless, antibodies against DNA and RNA
can be produced by stabilizing and linking them to an immunogenic carrier. In fact, several
serious human autoimmune diseases, such as systemic lupus erythematosus, are the result of
autoantibodies to nucleic acids. The discussion below is limited to carbohydrate antigens.
Correlation between molecular alterations and tumor behavior is well established.
Tumor behavior is related to the products of changes in cancer-related genes. The study of
indirect gene products such as cell surface carbohydrates is important in understanding
malignancy because tumor development is usually associated with changes in these
carbohydrates. These changes are often divided into alterations related to terminal carbo-
hydrate structures (which include incomplete synthesis and modification of normally exist-
ing carbohydrates) and alterations in the carbohydrate core structure. The latter includes
chain elongation of both glycolipids and proteins, increased branching of carbohydrates in
N-linked glycoproteins, and blocked synthesis of carbohydrates in O-linked mucinlike
glycoproteins. The importance of studying such changes becomes obvious considering that
the expression of carbohydrate antigens increases in a number of epithelial malignancies,
including ovary, lung, bladder, breast, colon, prostate, and gastric carcinomas. In fact,
tumor-associated carbohydrate changes are being used in the diagnosis of human cancers,
and the expression of some carbohydrate structures is associated with prognosis.
205
206 Chapter 9
Ovarian Carcinoma
Ovarian epithelial tumors, the most common ovarian malignancy, are usually categorized
into four main types: serous, mucinous, endometrioid, and clear cell. The tumors are fur-
ther classified as benign, of low malignant potential (borderline), or malignant, which are
further differentiated into four grades. The histological patterns, especially of malignant
tumors, may be a mixture of varying proportions of different histological types. Ovarian
carcinoma is often asymptomatic in its early stages. Approximately two-thirds of the
patients are diagnosed with stage III and IV disease, when metastatic spread is present
within the peritoneal cavity and/or to distant organs. Serous carcinoma is the most serious
and common histological subtype of ovarian carcinoma.
Other Applications of Microwave Heating 207
Both protein antigens and carbohydrate antigens can be retrieved in formalin-fixed and
paraffin-embedded tissues using enzyme digestion pretreatment, although the former gen-
erally are better retrieved by heat pretreatment. Guhl et al. (1998) have achieved enhanced
specificity and intensity of immunogold labeling of sugar moieties (poly a 2, 8 KDN
glycotope of megalin) present on O-glycosidically linked oligosaccharides by pretreating
the sections with N-glycanase F. This treatment also augments immunogold labeling of
certain membrane proteins in thin cryosections at pH 5 to 6.
The mechanism responsible for improved detection is thought to be better accessibil-
ity of glycotopes to the antibody; glycotopes usually are masked by unrelated large
oligosaccharides. The enzyme treatment eliminates steric hindrance by these oligosaccha-
rides. The mechanism of antigen retrieval essentially is based on the depolymerization of
Other Applications of Microwave Heating 209
The metaphase nucleolar organizer regions (NORs) are chromosomal segments or loops
(rDNA) containing ribosomal genes associated with proteins such as upstream binding factor
and RNA polymerase 1. These genes are clustered in 10 loci of the human acrocentric chro-
mosomes 13, 14, 15, 21, and 22. The transcriptional activity of these intranuclear segments
plays a pivotal role in the formation of nucleoli, directing the synthesis of both ribosomes and
associated proteins. The set of proteins associated with NORs are an acidic, nonhistone type
that binds silver ions and that are selectively stained. Silver binds with those rDNA sites that
are transcriptionally active or have already been transcribed and still retain residual rRNA
non-histone-associated proteins. The NORs stained with silver and the argyrophilic NOR-
associated proteins are called AgNORs and AgNOR-associated proteins, respectively.
The AgNOR proteins appear as distinct black structures under the light microscope.
Three different AgNOR staining patterns of metaphase chromosomes in human lympho-
cytes have been identified (Héliot et al., 2000): pair, sticklike, and unstained structures.
Chromosomes 13, 14, and 21 carry predominantly pair or sticklike AgNOR structures,
while 15 and 22 carry mainly pair AgNOR structures or remain unstained. Different
AgNOR shapes represent both the number of ribosomal genes carried by each chromo-
some and the differential recruitment of active ribosomal genes in each NOR cluster.
In interphase cells, the silver-stained structures are exclusively located within nucleoli
(Fig. 9.2). At the ultrastructural level each silver-stained structure corresponds to a fibril-
lar center with a closely associated dense fibrillar component (Derenzini et al., 1990).
Silver-stained NOR-associated proteins play a key role in the control of ribosomal
RNA (rRNA) transcription and processing and are considered markers of active ribosomal
genes. The close relationship between the rate of cell proliferation and ribosomal biogen-
esis is well established. In fact, a linear correlation exists between AgNOR counts and the
growth fraction in various malignancies, including carcinoma of the breast (Öfner et al.,
1996). For example, AgNOR parameters correlate significantly with MIB-1 growth frac-
tion and p53 protein expression (Bànkfalvi et al., 1998). AgNOR expression is markedly
higher in cycling (MIB-1 positive) tumor cells than in resting (MIB-1 negative) ones.
However, the AgNOR size rather than its number may correlate positively with elevated
proliferative status. Cumulative evidence indicates that malignant cells frequently exhibit
more AgNOR protein compared with that in benign or normal cells. Pich et al. (2000) have
presented a list of 29 tumor types for which AgNOR protein quantity is a prognostic factor.
Silver staining of NORs is influenced by the fixative, temperature, and duration of
staining. In general, the higher the temperature, the shorter the time required to stain
NORs. Prolonged staining results in nonspecific staining, and if staining is excessive, the
whole nucleus may appear homogeneously stained with silver. Considering the numerous
variables mentioned above and others that may influence NOR stainability, disagreements
over interpreting staining results in different laboratories are inevitable. Therefore, stan-
dardization of the preparatory protocols is necessary to obtain reproducible results. The
standardized silver staining procedure consists of heating the sections in a microwave
210 Chapter 9
Other Applications of Microwave Heating 211
oven, an autoclave, or a pressure cooker. Section staining is carried out in the dark at 37°C
using prewarmed solutions.
Two methods are available for the quantitative analysis of AgNOR proteins: the count-
ing technique and the morphometric method (Trerè, 2000). The counting technique consists
of enumerating each silver-stained dot per cell under the light microscope at a magnification
of 100X. The limitation of this technique is that when single AgNOR dots are clustered
together or partially overlapped, it becomes subjective and poorly reproducible. Further-
more, the counting technique does not take into consideration the size of each
silver-stained dot, which is variable. Clustering of dots and dimensional variability of the
dots are common in rapidly proliferating cancer cells. Another disadvantage of this technique
is significant interobserver variation. In addition, the technique may fail to demonstrate any
prognostic relevance of the AgNOR number to neoplastic diseases, including colorectal
carcinoma and breast cancer (Hennigan et al., 1994; Toikkanen and Joensuu, 1993).
To circumvent the above limitations, the morphometric method can be used (Rüschoff
et al., 1990). It consists of automatic or semiquantitative measurement of the area occu-
pied by the silver-stained structures within the nuclear profile with computer-assisted
image analysis (Trerè et al., 1995). This method is faster, more accurate and reproducible,
and shows less interobserver variation. Moreover, in contrast to the counting technique, the
morphometric method is predictive of patient survival, independent of the clinical stage of
the disease (Öfner et al., 1995a,b).
The morphometric method can be carried out with a CCD camera mounted on a light
microscope and connected to a personal computer equipped with specific morphometric
software. For example, Leica Quantimet SOOC image analyzer and processing system can
be used. A JVC TK-1280E videocamera, connected to a Leitz Orthoplan light microscope,
is used to record the images. QWIN VO1.00 software (Leica) can be used. The area of
the nucleus and of each AgNOR, the total area of AgNORs, and the area ratio of AgNORs/
nucleus (AR) are calculated automatically, together with AgNOR length, breadth, perime-
ter, roundness, and the aspect ratio of each nucleus (Staibano et al., 1998). Values are
expressed in micrometers.
Procedure
The following procedure is more suitable for routine application than other methods;
as many as 200 specimens can be processed at a time with this procedure. Sections
thick) of formalin-fixed and paraffin-embedded tissues are mounted on silane-coated
slides. They are deparaffinized with xylene and rehydrated in a series of descending con-
centrations of ethanol. The sections are immersed in 0.01 M sodium citrate buffer (pH 6.0)
in plastic Coplin jars and heated in an autoclave at 120°C for 20min. After the sections
have cooled down to room temperature for 20 min, they are incubated in the freshly pre-
pared following silver staining solution for 25 min at room temperature.
The sections are thoroughly rinsed in deionized water to remove unwanted silver precipi-
tates, dehydrated in a series of ascending concentrations of ethanol, cleared in xylene, and
212 Chapter 9
mounted. A second set of sections are stained without autoclave pretreatment. The results
of this procedure are shown in Figure 9.3.
Nucleolar Size
although nucleolar hypertrophy also occurs in normal proliferating cells. Thus, the value
of changes in nucleolar size in tumor pathology has been questioned. However, the rela-
tionship between nucleolar function as well as nucleolar size and cell doubling time indi-
cates the importance of the nucleolus in tumor pathology. Also, it has been shown that in
cancer cell lines characterized by different proliferation rates, the transcriptional activity
of RNA polymerase 1 and the expression of the major nucleolar proteins involved in the
control of rRNA transcription and processing (e.g., RNA polymerase 1, nucleolin, fibril-
larin, and protein B23) are directly related to nucleolar size and the rapidity of cell prolif-
eration (Derenzini et al., 1998).
To evaluate nucleolar size, histological sections are silver-stained for AgNOR
proteins. This procedure facilitates clear visualization of nuclear structure and size that can
be precisely measured by computer-assisted morphometric analysis (Öfner et al., 1995a).
Nucleolar size reliably indicates the rapidity of cell proliferation, inferring the prolifera-
tion rate of cancer cells. Higher AgNOR protein values correspond to worse clinical
outcomes. Fast-growing tumors have greater rRNA transcriptional activity than slowly
growing ones (Derenzini et al., 2000). Therefore, the shorter the cell cycle, the greater is
the rRNA transcriptional activity per unit of time and the greater the nucleolar size. This
conclusion is logical because proliferating cells must synthesize an adequate ribosomal
complement for the daughter cells.
IN SITU HYBRIDIZATION
The in situ hybridization (ISH) technique was introduced in the late 1960s, opening
a new era in histology and cell biology (Gall and Pardue, 1969). The technique was orig-
inally applied for localizing specific DNA sequences on chromosomes or interphase
nuclei. Interphase cytogenetics can be a very useful tool, for example, for bridging the gap
between cell culture and histology in tumor cytogenetics. The method is also important for
localizing specific mRNA sequences within cells and tissues. It allows the study of chro-
mosomal aberrations in routinely processed tissues. The overall advantage of ISH is that
by recognizing specific DNA or RNA sequences in a tissue or a cell, the precise location
of a potential or an effective synthesis of a given molecule can be determined. On the other
hand, immunocytochemistry demonstrates only the presence of protein molecules after
they have been synthesized.
In situ hybridization was derived from the techniques of molecular hybridization of
nucleic acids that are isolated from a particular cell population or tissue and bound to solid
supports. Hybridization of such averaged membrane-bound nucleic acids identifies differ-
ent classes of DNA (Southern blot) and RNA (Northern blot). However, ISH fills the gap
between the detection of a specific sequence and its precise location within the tissue or
the cell (Chevalier et al., 1997).
Literally hundreds of different hybridizations can be accomplished because ISH is often
carried out using semithin or thin sections of single tissue specimen (e.g., surgical biopsy),
using light and electron microscopy, respectively. A single specimen, on the other hand, is
often insufficient for Northern or Southern blot analysis. Another advantage of ISH is that it
allows the establishment of libraries of paraffin- or resin-embedded or frozen tissues. It has
been demonstrated, for example, that the hybridization signal can be maintained in frozen
sections stored at –70°C with a dessicant for more than 6 years (Wilcox, 1993).
214 Chapter 9
temperatures may disrupt the nucleocaspid protein coat of the measles virus, exposing
more nucleic acid for in situ hybridization (Shapshak et al., 1985). Another possible expla-
nation is that heating denatures the self-hybrids formed by folding of an mRNA molecule
or pseudohybrids formed between neighboring mRNA molecules in tissue sections
(Sibony et al., 1995). Heating may loosen these single mRNA strands, enhancing the
in situ hybridization efficiency.
The role of high temperatures in the breakdown of protein crosslinks introduced by
aldehydes has been discussed earlier in this book. The aforementioned combined tech-
nique is especially effective in increasing the in situ hybridization signal in archival tissues,
the fixation history of which may or may not be known. This protocol also detects low
levels of nucleic acids in the tissue, which may not be detectable with heating or enzyme
digestion alone.
In some cases, the use of medium wattages (e.g., 450 W) is preferred over high-power
microwave outputs (e.g., 700 W). This difference has been demonstrated in the ISH for
detecting measles virus and chicken anemia virus in formalin-fixed, paraffin-embedded
brain tissue (McMahon and McQuaid, 1996). In this study higher power outputs resulted
in decreased sensitivity. Although the exact reason for this phenomenon is not known, it is
hypothesized that optimal oscillation of dipolar molecules produces optimal thermal
effects in tissue sections at medium wattages.
Urea (0.01 M), sodium carbonate (0.01 M), magnesium chloride (0.01 M), or distilled
water can be used as microwave fluids to obtain similar results in terms of both sensitivity
and intensity of the hybridization signal. Alternatively, 10 mM citrate buffer (pH 6.0) can
be used as the microwave fluid. The major role of these fluids is to mediate high temper-
ature effects, which is confirmed by the achievement of a good hybridization signal using
distilled water. Note that pretreatment conditions must be optimized for every tissue type
and for every cell type in a given section.
Radioactive Probes
Radioactively labeled DNA and RNA probes as originally used (Gall and Pardue,
1969) are still widely applied for ISH because of their high sensitivity and strong amplifi-
cation of autoradiography. Also, radioactive probes enter tissue sections relatively easily.
Signal detection can be achieved within weeks with probes, while
probes yield autoradiographs within days. The disadvantages of these probes include
safety problems, reduced stability of radioactively labeled probes, and long durations of
exposure. These and other reasons stimulated interest in the development of nonradioac-
tive probes such as biotin and digoxigenin discussed below.
Nonradioactive Probes
Biotin is a small vitamin molecule ( 244) that binds with high affinity
to avidin. Avidin is a larger glycoprotein molecule ( 70,000), mostly distributed in egg
whites. This protein has the advantage of conjugating with different markers, including
peroxidase, fluorescent dyes, colloidal gold, and ferritin. Because of this property it is
216 Chapter 9
During this step, biotin sites on bound tyramine act as additional binding sites for antibi-
otin enzyme. An additional round of signal amplification can be achieved by using
biotinyltyramide and streptavidin conjugated to alkaline phosphatase (Yang et al., 1999).
Essentially, the CARD protocol is based on the deposition of haptenized tyramide
molecules in the vicinity of hybridized probes catalyzed by horseradish peroxidase. The
success of this technique depends on the integrity of target mRNA in sections and the abil-
ity of the probe to penetrate the sections and hybridize with mRNAs. Another requirement
is an efficient reporter system capable of revealing low numbers of probe-mRNA hybrids
per cell accompanied by low background staining.
Another approach to improve the resolution and detection sensitivity of the hybridiza-
tion involves the use of semithin resin sections instead of paraffin sections
or frozen sections. The former shows better preservation of cytological details
and higher spatial resolution. However, resin sections inhibit probe penetration into the
section, limiting probe hybridization to the target sequences/antibodies protruding from
the section surface. Such sensitivity and probe penetration difficulties can be circumvented
by using the methacrylate embedding–acetone deembedding (MEADE) technique (Warren
et al., 1998). These authors successfully localized mRNA and rRNA transcripts in marine
bivalves. Other resins such as LR White and Lowicryl K4M do not allow tissue deembed-
ding. Since this method requires longer incubation durations in acetone (12–15min) and
also a brief proteolytic digestion of the tissue to optimize intensity of the hybridization
signal, such treatments tend to have an adverse effect on cell morphology.
1. Fix human tissues with 4% paraformaldehyde in PBS for 24 hr, embed in paraf-
fin, and cut 4- to sections.
2. Mount sections on silane-treated glass slides.
3. Deparaffinize with three changes of 5min each in xylene, followed by three
changes of 100% ethanol, two changes of 95% ethanol, and two changes of 75%
ethanol.
4. Rinse in 0.85% NaCl.
5. Place slides in microwave transparent jars containing 0.01 M sodium citrate
buffer (pH 6.0), and heat to the boiling point at maximum power (700 W) in a
microwave oven equipped with a rotating plate; cover the jars with loosely fitting
lids unless 2–3 cm empty space is present above the buffer level in the jar.
6. Allow boiling to continue for 7 min.
7. Check the buffer level, add fresh distilled water if necessary, and again boil
for 5 min.
8. Cool the slides to room temperature for 20 min.
9. Rinse for 5 min in PBS (0.145M NaCl and 0.01M sodium phosphate buffer;
pH 7.3).
10. Postfix with 4% paraformaldehyde for 20 min.
11. Rinse twice for 5 min each in PBS.
12. Digest with proteinase K for 20 min.
218 Chapter 9
adjusted to the respective tissue. Following washing twice for 5 min each in distilled water,
the slides are air-dried and treated on a hot plate for 30 min at 80°C.
Each section is covered with of freshly prepared hybridization solution:
Deionized formamide (65%)
2 × SSC (0.3 M NaCl and 0.03 M sodium citrate)
Dextran (10%)
Salmon sperm DNA
Biotinylated probe DNA
Sections are covered with cover-slips, sealed with rubber cement, denatured by heat-
ing at 78°C in a water bath for 10 min, and hybridized overnight at 37°C. The coverslips
are carefully removed by floating the slides in 2 × SSC, and the sections are washed twice
for 10 min each in a mixture of 2 × SSC and 50% formamide at 40°C, and then three times
in PBS. Endogenous peroxidase is blocked by incubation in 1% for 15 min.
The labeled DNA is detected by incubating the slides in PBS (10.4mM
3.16mM 150mM NaCl, pH 7.6) containing 1.5% normal horse serum for 10 min
at 37°C. The fluid is decanted, and a monoclonal mouse antibiotin antibody, diluted 1:100 in
PBS, is added for 30 min at 37°C. The sections are washed three times in PBS and then incu-
bated for 15 min at 37°C in a biotinylated goat antimouse antibody (Vector, Burlingame,
CA), diluted 1:1000 in PBS containing 1% BSA. After three washes in PBS, the sections are
covered with peroxidase-conjugated streptavidin ( in PBS) for 30 min at 37°C. The
sections are carefully washed twice in PBS, and DAB (0.5 mg/ml in 0.05 M Tris-HCl buffer,
pH 7.6, and 0.03% ) is added as the chromogen. The sections are counterstained with
hematoxylin, dehydrated, and mounted. The results of this method are shown in Figure 9.4.
Skeletal tissues are mineralized and so require decalcification to obtain clear mor-
phological details. Decalcifying reagents such as EDTA and HC1 have been traditionally
used at room temperature for processing such tissues for in situ hybridization. However,
these and other similar agents require long durations of treatment, resulting in damaged
cell morphology and reduced hybridization signals. It has been demonstrated that long
decalcification, for example with EDTA at room temperature, reduces hybridization sig-
nals, while the signals are preserved better after treatment with the same reagent at the
same temperature for short-term decalcification (Kaneko et al., 1998).
The above-mentioned limitations can be significantly minimized by using microwave
heating. It has been shown that decalcification with EDTA in a microwave oven reduces
the duration for decalcification, which in turn prevents the reduction of hybridization sig-
nals caused by long-term decalcification (Kaneko et al., 1998). Formic acid has also been
used in conjunction with microwave heating as a decalcifying agent and has been reported
to decalcify faster than EDTA (Callis and Sterchi, 1998). This treatment has not been
tested for in situ hybridization. For in situ hybridization, each consecutive microwave
decalcification in the presence of 20% EDTA for 2hr at 50°C for 3–4 days is recom-
mended. The rest of the time, the tissues are decalcified in 20% EDTA at room tempera-
ture. A temperature lower than 55°C would safely preserve cell morphology.
220 Chapter 9
Other Applications of Microwave Heating 221
Traditionally, processing of plant tissues for light microscopy required about 1 week.
Although procedures are now available that allow paraffin embedding in short durations
(Ruzin, 1999), anatomical preservation is less than excellent. To circumvent these prob-
lems, Schichnes et al. (1999) introduced an efficient protocol using microwave heating for
paraffin embedding and DIG-labeled (digoxigenin-UTP) knotted (kn) mRNA as a probe for
hybridizing mRNA from the knotted gene in the meristematic tissues. This approach
decreases the durations of fixation from 24 hr to 15 min, dehydration from 73 hr to l0min,
and infiltration from 96 hr to 3 hr. This procedure also minimizes the time required for sec-
tion adhesion to slides as well as completion of staining. Moreover, anatomical preservation
is superior, and localization of the mRNA probe is precise. This protocol is detailed below.
Microwave Treatment
Areas of high microwave flux are checked with a Pelco 36140 microwave bulb array
(Ted Pella). Specimens are not placed in areas indicated by illuminated bulbs. Vials con-
taining the specimens are placed in a cold tap water bath (50 ml) that is preheated to the
required temperature. The temperature is regulated by placing the microwave temperature
probe into a vial of the same solution that is present in the specimen vial. The built-in tem-
perature probe displays the specimen temperature on the oven front panel. The wire that
attaches the probe to the oven is submerged in the water to decrease the antenna effect.
An additional 400 ml of static water load is placed in the oven at an optimal position deter-
mined with the microwave bulb array. This water is changed between every step.
Staining
Tissue sections ( thick) are placed on Probe-on Plus slides and floated on auto-
claved water on a hot plate (42°C) for 2–3 min to remove compression. The water is
removed with a paper towel, and the slides are placed on a slotted glass staining dish on
its side in the microwave oven. To adhere the sections, the slides are heated in a microwave
222 Chapter 9
oven at 43°C for 30 min. The probe is placed in a drop of water on a slide on top of the
slide rack to measure the temperature.
The slides are placed in the staining dish which is filled with 1% safranin O, in 1 part
methyl cellusolve, 1 part 50% ethanol, 1% (w/v) sodium acetate, and 2% formalin solu-
tion, placed in a water bath in the microwave oven, and loosely covered with plastic wrap
to prevent splattering; tight-fitting covers must not be used. The temperature probe is
inserted into the staining dish through the wrap. The slides are stained for 45 min at 60°C
in the oven.
The DNA in the sections is denatured by treatment with 70% formamide/2 × SCC for
5 min at 80°C. Ten microliters of the probe solution (hybridization buffer: probe:
and distilled water: ) is placed on the slide and coverslipped. The slide is placed in a
microwave oven (2.45 GHz, 300 W) and heated for 3 sec at 2-sec intervals for a total of
15 min at 42°C. DAPI II (4,6-diamidine-2-phenylindol) (125 ng/ml) is used for nuclear
staining. The sections are promptly observed under a fluorescent microscope equipped
with epifluorescence filters and a photometric CCD camera. The captured images are
digitized and stored in an image analysis program.
The number of signals per cell is counted for a total of 100–200 cell nuclei, and the
signal intensities of the different periods of hybridization are simultaneously compared.
To avoid the limitations of enzyme digestion, microwave heating alone can be used
for in situ hybridization. Microwave boiling facilitates the probe’s penetration through the
tissue to the nucleus. Also, this treatment causes at least partial denaturation of the nuclei,
enhancing hybridization. Microwave heating alone has been employed for fluorescence
in situ hybridization for chromosomal sequences (Bull and Harnden, 1999). The pretreat-
ment time is reduced to ~ 1 hr. Prostate tissue is fixed overnight with 4.4% formaldehyde
containing 1 % NaCl (pH 6.3). Paraffin sections ( thick) are placed on slides which can
be stored at room temperature.
The sections are deparaffinized, followed by hybridization. The slides are placed in
a glass staining jar containing a few antibumping granules (BDH Lab Supplies, Poole,
England), filled to the brim with a mixture of 100 mM Tris-HCl buffer and 50 mM EDTA
(pH 7.0), and placed at the center of a rotary 800 W microwave oven. Microwave boiling
is carried out at full power for 2–3 min. For four cycles of microwave heating, hot fluid
(65°C) is used to refill the staining jar as rapidly as possible to avoid drying of the slides.
The slides are transferred to 70% ethanol at 4°C and then to 100% ethanol at the same
temperature, followed by air-drying.
Digoxigenin-labeled chromosome 10 probe (DIOZI; Oncor, Gaithersburg,
MD) is used at a final concentration of in hybridization buffer (50% formamide,
10% dextran sulfate, 0.004% Tween 20, and standard saline citrate (SSC) [in 1.5 strength]).
A volume of the probe is placed on an 18 × 18-mm coverslip, which is placed onto
the slide, sealed with special Vulcanizing Fluid, and placed on a flat-bed thermal cycler.
Slides are incubated for 3 min at 94°C and placed in a humidified box for 16 hr at 37°C.
Coverslips are removed, and slides are placed in wash buffer (4 × SSC/0.05% Triton
X-100) twice for 2 min each. This is followed by a thorough wash in 0.25 × SSC for 5 min
at 72°C, transfer to wash buffer at room temperature, and flooding with blocking buffer
(0.5% milk powder in wash buffer) for 5 min. The signal is detected for 30 min at 37°C
with antidigoxigenin, diluted 1:100 in blocking buffer. Coverslips are gently removed,
and slides are rinsed three times for 2 min each in wash buffer at 45°C before mounting
in antifade (Vectashield, Vector Lab., Burlingame, CA) containing propidium iodide
Slides are viewed on an Axioskop microscope (Carl Zeiss) equipped with a
standard camera and appropriate software (PSI, League City, TX).
224 Chapter 9
The polymerase chain reaction (PCR) technology yields a DNA fragment for cloning
and is especially useful for cloning cDNAs. This technique uses the enzyme DNA poly-
merase to make a copy of a defined region of DNA. The region of the DNA we want to
amplify is selected by putting in short pieces of DNA primers that hybridize to DNA
sequences on either side of the selected region and cause initiation (priming) of DNA syn-
thesis through that region. The copies of both strands of the selected region, as well as the
original DNA strands, serve as templates for the next round of amplification. Thus, the
amount of the selected DNA region doubles again and again with each cycle.
The PCR technique can be used to study DNA from a variety of sources, including
archival specimens containing formalin-fixed, paraffin-embedded tissues (Alcock et al.,
1999). Microdissection can be performed on sections cut from such tissues that have been
processed for conventional immunohistochemistry. Crude DNA extracts, obtained from
microdissected specimens by microwave heating, can be added directly to amplification
reactions. Analyses using a range of PCR-based techniques, including microsatellite repeat
polymorphism analysis at the NM23-H1 locus and sequencing of exon 5, 7, and 8 of the
p53 gene, can be performed.
Procedure
Flow cytometry has been a versatile method for detecting and quantifying cell surface
antigens in diagnostic and research fields for many years. Recently, it has also become use-
ful for simultaneous detection of cytoplasmic and nuclear antigens by using optimal fixa-
tion and cell permeabilization protocols. Generally, cell fixation and cell membrane
permeabilization are mandatory for the detection of intracellular antigens. Fixation can be
carried out with an alcohol or an aldehyde, and cell membrane permeabilization can be
accomplished by treating the cells with detergents such as Tween 20, Triton X-100, saponin,
lysolecithin, or NP-40. Detergents facilitate access of large molecules such as antibodies
and DNA fluorochromes to the target antigen after or before the cells have been fixed.
Care is required to achieve optimal cell membrane permeabilization. If permeabiliza-
tion is insufficient, the accessibility of antibodies to the target antigen will be hampered,
resulting in underscoring (Lan et al., 1996). If permeabilization is excessive, cell mor-
phology will be damaged, causing a nondiscrimination pattern in cell populations.
Consequently, it is difficult to use light scatter signals to gate on the cells for accurate
analysis. Since optimal fixation and detergent permeabilization depend on the type of cell
and the characteristics of the antigen under study, these parameters are determined by trial
and error. Thus, this approach is somewhat tedious and time consuming.
Image cytometric quantitation of nuclear immunostaining can be carried out with the
CAS 200 image cytometer (Becton Dickinson, San Jose, CA). It is based on differential
staining of nuclei for the antigen (e.g., Ki-67 in ductal carcinoma of the breast using
microwave heating and MIB-1 antibody) (Bhoola et al., 1999). Immunopositive nuclei are
brown, while negative nuclei counterstain as blue with hematoxylin. Using the two CAS
200 sensors, measurements are made at different wave lengths. At 620 nm, both brown and
blue absorb, providing a mask of all nuclear material. At 500 nm only brown stain absorbs,
allowing the positive nuclei to be measured independently. Comparison with the 620-nm
mask gives a percentage of nuclei area stained positively.
The image cytometer is standardized by adjusting the light source of the microscope
to a predetermined value on an empty field. Then, in control mode and on the negative con-
trol slide, the antibody threshold is adjusted using nuclear areas considered negative. After
comparing the brown mask with the blue and the brown-stained image seen through the
microscope, the slide stained for the antigen is analyzed in the specimen mode.
Fifteen high-power fields are analyzed in each case, using random but consecutive
fields when possible. Tumor cells are isolated from stroma by using the scene segmenta-
tion function, which allows the operator to precisely define portions of the image to be ana-
lyzed (Bhoola et al., 1999). Computer-generated histograms show the percentage of
positive nuclear area on the vertical axis and nuclear optical density on the horizontal axis.
It is thought that the microwave heating method is comparable to the OPF and F&P pro-
tocols, though the former method is inexpensive, which is important because in a diagnostic
laboratory cost is a consideration in choosing a particular method. Moreover, the exact ingre-
dients of OPF and F&P are not known. It should be noted, however, that unlike these com-
mercial reagents, microwave heating may damage or cleave sensitive cell surface antigens,
preventing cell selection on the basis of combined cell membrane and intracellular antigens,
but microwaving decreases nonspecific background fluorescence (Millard et al., 1998).
Procedure 1
Cells are washed in PBS and fixed with 2% paraformaldehyde for 30 min at
4°C. The supernatant is discarded after centrifugation (500 g for 5 min at room tempera-
ture), and cells are resuspended in 10 ml of 0.01 M sodium citrate buffer (pH 6.0) contain-
ing 0.5% bovine serum albumin in an unsealed 50-ml propylene tube. The tube is placed
upright in the center of a 1-liter Pyrex glass beaker, which is sealed with polyethylene plas-
tic wrapping. The cell suspension is heated for 30–60 sec in a microwave oven at the max-
imum power setting (800 W), reaching a temperature between 90 and 100°C. The cells are
chilled on ice for 10 min. After centrifugation at 500 g for 5 min, the supernatant is dis-
carded and the cells are washed in PBS. The cells can be filtered through a mesh
to remove the aggregated debris and then labeled with monoclonal antibodies for flow
cytometry as described below.
The cells are incubated in the primary antibody at an appropriate dilution for
30–60min at 4°C or room temperature, depending on the type of cells or antigens. After
being washed in PBS, the cells are incubated in a fluorescein isothiocyanate (FITC)–con-
jugated goat antimouse IgG (or sheep antimouse IgG) for ~30 min at 4°C in the dark. The
cells are washed twice in PBS and resuspended in of 1% fetal calf serum or
ISOTON II for flow cytometric analysis.
The cells are run on a flow cytometer, an EPICS 752 (Coulter Electronics, FL) con-
nected to a CICERO data acquisition system (Cytomation, CO) or FACScan (Bectin
Dickinson). An argon ion laser (Coherent, CA) operating at 488 nm is used to illuminate
the cells. Forward and right-angle light scatter signals are collected along with FITC fluo-
rescence (measured through a 535-nm bandpass and logarthimic amplification) and PI flu-
orescence (630 nm long-pass filter) where appropriate. Fluorescence histograms of at least
5,000 counts are generated from a gate set in the forward angle versus 90°C light scatter
scattergram. The percentage of positive cells is measured from a cutoff set using an
isotype-matched, nonspecific control antibody, while the mean channel fluorescence is
measured over the entire distribution. Figure 9.5 shows clear discrimination between intact
cells and cell debris after microwave heating.
Although the above method is highly recommended, if the expression of immuno-
staining is weak because of antigen masking and inaccessibility of antigens to antibodies
in the aldehyde-fixed and paraffin-embedded tissues, single cell/nuclei can be isolated
from archival paraffin-embedded tumors for laser flow cytometry using fluorescent-
labeled primary or secondary antibodies. This approach is especially useful for steroid hor-
mones such as estrogen and progesterone, which have nuclear binding sites. The advantage
of nuclear isolation is the greater accessibility of immunoreagents to the nuclear proteins
compared with that in the nuclei of whole cells.
228 Chapter 9
Another advantage is the recognition of two subpopulations with low and high stain-
ing. Such a heterogeneity of the nuclear antigenic expression (e.g., estrogen) is not seen in
the whole cell preparation analyzed by flow cytometry. This heterogeneity is possibly due
to improved reactivity and maximum access of the nuclear proteins to the antibodies.
Heterogeneity of nuclear protein expression is thought to be due to the variations in the
content of nuclear proteins, their cell cycle stage, and proliferation (Sabe et al., 1999). In
this respect, different physiological states and differences in size and surface charge of the
protein are also important.
The nuclear isolation method has been used for processing archival paraffin-embedded
mammary tumors for monitoring estrogen expression and aneuploidy (Sabe et al., 1999).
These two parameters have important diagnostic and prognostic significance in mammary
tumors.
Procedure 2
Approximately sections cut from formalin-fixed and paraffin-embedded tis-
sues (e.g., breast) are treated with 0.05% pepsin (cat. no. P7012, Sigma) in normal saline (pH
1.65) for 1 hr at 40°C (Sabe et al., 1999). The sections are vortexed every 5 min for an additional
30 min. This proteolytic reaction is terminated by adding 5 ml of cold 10% fetal bovine serum
(FBS) in ethylene-diaminetetraacetic acid (EDTA). After filtration through nylon mesh,
the filtrate is forced through a syringe with a 28-gauge needle and centrifuged at 300g for
7 min. The resulting pellet is resuspended in 1ml of nuclear isolation medium (Hank's PBS
with 0.2% FBS, 25 mM HEPES buffer, and 0.6% NP-40) for l0 min at 4°C.
The cells and nuclei are aliquoted into polystyrene tubes. Approxi-
mately of normal horse serum (Vector Laboratories, Burlingame, CA) is added to
block any nonspecific binding. The suspension is incubated with biotinylated antiestrogen
monoclonal antibody (1D5, Dako Corp., Carpinteria, CA) at 1:25 dilution for 1 hr at 37°C.
Aliquots are stained with of fluorescein isothiocyanate (FITC)–conjugated strepta-
vidin (Dako buffer containing 0.1% Triton X-100).
The suspensions are centrifuged and washed twice in 3% FBS/PBS. They are again
centrifuged and washed twice in 3% FBS in PBS with 0.1% Triton X-100. The pellets are
resuspended and incubated with 0.5 ml of 1% FBS containing propidium iodide (
Calbiochem, San Diego, CA) and RNAse (1 mg/ml) in Hank’s balanced salt solution (with-
out phenol red) for 30 min at 37°C. The samples are analyzed on a Coulter Electronics
XL-MCL or a Becton Dickinson FACScan flow cytometer with standard argon ion laser
excitation and filter configuration for the FITC/propidium iodide dye combination.
Like other specimens, bacteria can be processed for scanning electron microscopy
(SEM) using microwave heating. Conventional processing of specimens for SEM is car-
ried out in ~4 hr, while they can be prepared in ~1 hr using microwave heating (Fox and
Demaree, 1999). Bacterial cells at an early exponential growth phase on polycarbonate
membrane filter (Nucleopore Corporation, Pleasanton, CA) can be used. The membrane
filter with attached cells is cut into small squares (~50 × 50 mm) and transferred to
polypropylene Petri dishes (60 × 15 mm), which are then placed in the cold spots in the
microwave oven at power level four (536 W). Previous to this step, using the neon bulb
array, cold spot determination has been done. Also, two beakers containing water have
been placed in the microwave oven as water loads to absorb microwave energy. The fixa-
tion is accomplished by placing ~ 1–2 ml of for 20 sec in the Petri dish.
The temperature probe is placed into a blank polypropylene Petri dish during pro-
cessing, and the temperature is restricted to 37°C to prevent overheating. The filtrate is
rinsed three times for 5 min each with 1–2 ml of phosphate buffer. The sample is dehy-
drated in a microwave oven with 1–2 ml of an ethanol series of increasing concentrations
(once in each of 50%, 70%, and 90% ethanol and three times in 100% ethanol). Each dehy-
dration step is carried out for 20 sec in the microwave oven at power level 4 (536 W) with
a temperature restriction of 37°C utilizing the temperature probe placed in the blank Petri
dish containing ethanol.
The filtrate is further dehydrated in 100% hexamethyldisilazine (HMDS) for 20 sec at
37°C in a microwave oven at the same power level utilizing the temperature probe in the
blank Petri dish containing HMDS. The filtrate is dried in a conventional oven for 15 min at
60°C. After 15 min all excess HMDS is removed, and the filter with attached cells is allowed
230 Chapter 9
to continue to dry in the oven. The samples are sputter-coated with gold (1.5 min at 40 mA)
and then viewed in a scanning electron microscope. The whole process takes ~ 1 hr. The
results of this procedure, as shown in Figure 1.2, are similar to those obtained with stan-
dard technique. For additional information on the microwave heat–assisted processing of
specimens for scanning electron microscopy, the reader is referred to Demaree (2001).
Confocal laser scanning microscopy can be used in conjunction with microwave heating
for examining the three-dimensional structure and cellular interrelationships in sections of
paraffin-embedded tissues (Boon and Kok, 1994). Tissues are fixed with Kryofix, a coag-
ulant fixative containing 50% ethyl alcohol and polyethylene glycol (PEG; molecular
weight 300) for 90 sec in a microwave oven. The use of thick paraffin sections and
fluorescently labeled antibodies is preferred.
The choice of tissue processing method is crucial for optimal detection and quantifi-
cation of target antigens in cells by immunogold light and electron microscopy. The primary
criteria for choosing a method are efficient, specific, and reproducible labeling of the antigen
and satisfactory preservation of cell morphology. In some cases correlative light and
electron microscopy for analyzing immunostaining is desirable. These objectives can be
achieved by observing semithin and thin sections of the tissue embedded in water-miscible
(e.g., Lowicryl) or water-immiscible (epoxy resins) media. Semithin sections allow a sur-
vey of the spatial distribution of the antigen, and thin sections provide subcellular expres-
sion of the antigen on consecutive sections. Immunostaining of both semithin and thin
epoxy sections can be enhanced by controlled etching of sections with sodium ethoxide
(0.6% hydrogen peroxide in 96% ethanol) followed by antigen retrieval in a microwave
oven. This procedure was recently used for immunostaining of E-cadherin, and
in the human proximal jejunum (Groos et al., 2001).
One of the advantages of using an epoxy resin is that, in contrast to cryosections, each
tissue block can be repeatedly sectioned for both light and electron microscopy. Treatment
with ethoxide permeabilizes the section surface by partial corrosion of the embedding
resin. It is essential to determine the optimal concentration of the etching agent and etch-
ing duration to obtain sufficient permeability of the section surface for antibody access
while avoiding structural damage. It is also necessary to find out optimal heating treatment
for unmasking antigens hidden by covalent bonds formed between epoxy resin and bio-
logical material during polymerization.
Procedure
Some cell-proliferating antigens that are detected with antigen retrieval methods using
frozen or formalin-fixed and paraffin-embedded tissues are discussed below. They include
cell nuclear proliferating antigens (Ki-67 and proliferating cell nuclear antigen [PCNA]),
p53, estrogen, androgen, and progesterone. Such immunohistochemical studies are important
for diagnostic, prognostic, and therapeutic purposes. These studies are in common use to
assess the importance of several antigens as prognostic factors in all kinds of malignan-
cies. This approach is more commonly used in pathology laboratories than is analysis of
gene mutation at the molecular genetic level, which is cumbersome and time consuming.
Before discussing cell nuclear proliferating antigens, it is relevant to briefly explain the
cell cycle. Proliferating cells can occupy several functional states besides mitosis. After com-
pleting mitosis, the daughter cells enter the Gap 1 phase. The duration of phase varies
with the tissue type. Subsequently, cells enter the S (synthetic) phase, where the cell’s genetic
material is doubled during DNA synthesis. This phase is followed by a second Gap
phase before cells divide again. The durations of these phases in descending order are
(8–10 hr), S (6–8 hr), (4–6 hr), and mitosis (30–45 min). Proliferating cells after the
phase leave the cell cycle, cease proliferation, differentiate, and eventually die or they enter
a resting phase from which they may be recruited back into the cell cycle at a later time.
KI-67 ANTIGEN
Ki-67 is a highly positively charged alkaline nonhistone protein (pI = 9.9) with repet-
itive elements and a high content of randomly distributed prolines (8.2%) and lysines
(11.4%) (Duchrow et al., 1994). It is encoded by a single gene on chromosome 10. The
protein is a bimolecular complex of molecular weight 345 and 395 kDa. Ki-67 monoclonal
antibody detects two polypeptides of these molecular weights in proliferating cells. Since
Ki-67 protein contains many proline–glutamic acid–serine–threonine (PEST) motifs, this
protein is capable of being very rapidly catabolized (Rogers et al., 1986). Thus, it has a
half-life of only 1–2 hr. Cloning and sequencing of the complete cDNA of Ki-67 antigen
have been carried out (Duchrow et al., 1994).
The Ki-67 antigen was originally identified by its cell-cycle-related expression and is
now considered to be a more specific marker for cell proliferation and cell cycling than is
PCNA (Gerdes et al., 1984). This is supported by the observation that ependymal cells,
233
234 Chapter 10
which are unable to regenerate, are PCNA-positive, but Ki-67-negative (Sarnat, 1995;
Funato et al., 1996).
Ki-67 antigen is expressed throughout all phases of the cell cycle ( S, and M),
except the quiescent phase. This means that the expression of this antigen is inti-
mately associated with the cell cycle. The topographical distribution of Ki-67 antigen is
also cell cycle-dependent. Its expression becomes apparent at the beginning of the
phase and accumulates within the nucleus (predominantly in the perinucleolar region) dur-
ing the late phase. The expression of this antigen increases as the cell cycle progresses,
reaches its maximum concentration at mitosis, and markedly diminishes thereafter. In fact,
immediately after mitosis its amount is minimal.
In the phase Ki-67 antigen is predominantly located in the nucleoli and is partic-
ularly evident in cells containing relatively large nucleoli. Treatment with DNase or RNase
indicates that the antigen located in the nucleoli is associated with RNA (Szekeres et al.,
1995; Benfares et al., 1996). In the later phases of the cell cycle it is also detected through-
out the nucleoplasm, being found mostly in the nuclear matrix, where it is associated with
DNA. During mitosis, it is present on all chromosomes and appears in a reticulate struc-
ture surrounding the metaphase chromosomes (Verheijen et al., 1989). The chromosomal
binding of Ki-67 antigen is considered to be due to electrostatic attraction. Because of the
short half-life of Ki-67, it is rapidly degraded, resulting in decreased immunostaining
during anaphase and telophase.
Ki-67 antigen is also sensitive to the nutritional status of cells, declining rapidly after
3 days of nutrient depletion in mitotic cells or becoming undetectable upon nutritional dep-
rivation in lung cancer cells (Verheijen et al., 1989; Tinnemans et al., 1995). However,
according to Dong et al. (1997), a greater relative change in Ki-67 expression occurs under
conditions where proliferation is inhibited without growth fraction change than under con-
ditions where a significant change in growth fraction does occur. It should be noted that
these two observations were made using different cell types.
It is apparent from the above discussion that Ki-67 is present in the nucleus of prolif-
erating cells and is an indicator of the growth fraction in tumor cells. It is primarily a DNA-
binding protein that plays a crucial role in the maintenance or regulation of cell division.
This protein may also function as a matrix for chromosomal DNA or contribute to the con-
densation of the chromosomes or be involved in breakdown of the nuclear membrane before
mitosis (Duchrow et al., 1994). The association of Ki-67 with RNA in the nucleoli and with
the DNA with nuclear matrix suggests that the antigen plays a role in transcriptional
processes as a structural protein by mediating between nuclear DNA and nucleolar RNA.
Ki-67 antigen is a valuable tool for measuring cell growth in human tissues and cell
cultures, particularly with respect to the histopathological determination of malignancy. It
can provide information on the fraction of actively cycling cells. In certain malignant
tumors the number of tumor cells immunohistochemically positive for Ki-67 antigen coin-
cides with estimated tumor proliferation rates (Gerdes et al., 1983). In fact, this antigen is
absolutely required for maintaining active cell proliferation. In addition, immunohisto-
chemical evidence indicates that Ki-67 antigen may be associated with neurofibrillary
degeneration in Alzheimer’s disease, other neurodegenerative disorders, normal aged
brains, and neoplasms such as gangliogliomas (Smith and Lippa, 1995). This antigen pos-
sibly plays a role in the production of abnormally phosphorylated tau protein, which leads
to the formation of paired helical filaments within susceptible neurons. Considering these
Cell Proliferating Antigens 235
functions, Ki-67 antigen is expected to be expressed in all active parts of the cell cycle.
Unlike PCNA, Ki-67 is not involved in DNA repair.
Ki-67 has a much shorter half-life (1–2 hr) than PCNA. Therefore, unlike the latter,
the former demonstrates the proliferative stage of the cell rather than the residual evidence
of the cell that has passed through the cell cycle stage. It has been demonstrated that nor-
mal tissues adjacent to carcinoma are PCNA-positive but Ki-67-negative (Wolf and
Dittrich, 1992). On the other hand, in an immunohistochemical study, Ki-67 expression
was of no predictive value in squamous cell carcinoma of the head and neck (Roland et al.,
1994). A recent study also indicates that the expression of Ki-67 in primary intraoral squa-
mous cell carcinomas of the head and neck is independent of tumor site (Nylander et al.,
1997). It has also been demonstrated that Ki-67 antigen staining might contribute to false
data on the growth fraction (Ansari et al., 1993). If this problem arises, it can be resolved
by employing double labeling with two markers, Ki-67 and statin, for proliferating cells
and resting cells, respectively. However, overwhelming evidence has established Ki-67
immunohistochemistry as an important tool for assessing cell proliferation in situ.
It should be noted that, like most other antigens, the detectability of Ki-67 is highly
influenced by fixation and other preparatory parameters.
Immunohistochemistry
can provide an objective and reproducible assessment (Biesterfeld et al., 1995). The pres-
ence of Ki-67 antigen can also be determined by flow cytometric methods with improved
reproducibility but at the cost of cell morphology details (Steck and El-Naggar, 1994).
Limitations of Immunohistochemistry
The labeling index (percentage of positively stained nuclei) is often found to vary
between fields within the same tumor specimen because of the heterogeneous distribution
of proliferating cells, which can introduce sampling error. Also, the values obtained for the
labeling index may vary among laboratories, depending on storage and handling proce-
dures, thus limiting the usefulness of a direct comparison of the labeling index values. In
this respect, interobserver and intraobserver detection variations in the same laboratory
also cannot be ignored.
Also note that the proliferation rate of the tissue depends not only on the number of
cells in the cell cycle but also on the time taken to complete a whole cell cycle and on
whether cells undergo programmed cell death. Because the Ki-67 labeling index measures
only the number of cells that are cycling and gives no indication of the time required for
the cell cycle, a tumor might be proliferating rapidly and still may show a low labeling
index, or be proliferating slowly but remain in stage and so have a high labeling index.
In other words, a tumor with many cells in a cycle can be strongly immunostained using
MIB-1 antibody even though the tumor has a slow cell cycle and a low proliferating rate
(Jansson and Sun, 1997). In contrast, a tumor with a short cell cycle and high proliferation
rate might not be stained since there are few cells in the cycle.
The above-mentioned possibility is one of the reasons for lack of association between
Ki-67 immunostaining and clinicopathological variables and prognosis, for example, in
colorectal carcinoma. For this and other reasons several studies have indicated that Ki-67
staining has very little prognostic value in gastric and colorectal carcinomas (Victorzon et al.,
1997; Jansson and Sun, 1997). However, in spite of these potential limitations, the assess-
ment of proliferation using the Ki-67 labeling index provides valuable prognostic informa-
tion in many tumor types, including lymphomas, gliomas, and breast tumors. Ki-67 labeling
index is considered to be a more objective way of predicting malignant transformation than
traditional histopathological evaluation alone.
Antibodies
Monoclonal antibodies Ki-67, MIB-1, Ki-S5, and MIB-5 recognize Ki-67 antigens
on sections of formalin-fixed and paraffin-embedded tissues. Using these antibodies in
conjunction with immunohistochemistry, a rapid and reproducible determination of the
growth fraction of a given human cell population can be accomplished. The determination
of the growth factor is an objective aid for defining the outcome of an individual tumor
case and is particularly useful for selecting appropriate individual tumor therapy.
The Ki-67 antibody was obtained in studies aimed at the production of monoclonal
antibodies to nuclear antigens specific to Hodgkin and Sternberg-Reed cells (Gerdes et al.,
1983). There is a highly significant correlation between the mean value of the growth
238 Chapter 10
fraction determined with this antibody and the histopathological grade of malignancy.
Immunostaining of Ki-67 antigen with Ki-67 antibody can be enhanced by exposing the
cells to increasing ionic strength (1.15–1.6 M NaCl) during fixation with paraformaldehyde,
suggesting increased accessibility of the antibody to the epitope (Bruno et al., 1992). Note
that epitope denaturation increases at higher ionic strengths. This antibody is used mostly
on frozen sections, although it can be employed on sections of fixed and paraffin-embedded
tissues after treatment with an antigen retrieval method such as microwave heating. However,
the immunoreactivity of this antibody tends to be inconsistent because the epitope in fresh
specimens may be lost during routine histopathological processing.
To circumvent the limitations of Ki-67 antibody, Ki-67 antibody equivalent murine
antibodies (MIB-1–3) were generated against bacterially expressed parts of the Ki-67 cDNA
containing three 62-base-pair repetitive elements encoding for the Ki-67 epitope (Key et al.,
1993). MIB-1 shows affinity with both native Ki-67 antigen and recombinant parts of the
antigen. MIB-1 has excellent immunostaining properties for Ki-67 antigen, not only in
frozen tissues but also in routinely fixed and paraffin-embedded specimens. In fact, MIB-1
exhibits an immunostaining pattern identical to that of Ki-67 antibody in fresh specimens.
This advantage of MIB-1 allows retrospective studies using archival specimens. The versa-
tility of MIB-1 as a marker of Ki-67 antigen in a wide variety of malignant neoplasms is
indicated on pages 39 and 239.
Ki-S5 is another antibody generated against the Ki-67 antigen to label a formalin-
resistant epitope in routinely processed tissues (Kreipe et al., 1993). Crude nuclear extracts
of the Hodgkin-derived cell line L428 were used for the immunization of mice and the pro-
duction of this antibody. The immunoreactivity of Ki-S5 antibody is confined to the nuclei
of proliferating cells and, unlike Ki-67 antibody, does not cross-react, for instance, with
cytoplasmic antigens of epithelial cells (Rudolph et al., 1995). Moreover, Ki-S5 antibody
yields identical results in fresh or fixed tissues. Parallel staining of Ki-67 and Ki-S5 anti-
gens using Ki-67 and Ki-S5 antibodies, respectively, yields almost identical results in non-
Hodgkin’s lymphoma (Kreipe et al., 1993). Retrospective studies relating the proliferative
activity to clinical outcome are rendered possible with antibody Ki-S5 using archival spec-
imens that have been fixed and embedded. Ki-S5 antibody is available free on request from
the Institute of Pathology, Kiel, Germany. MIB-5 is yet another antibody that recognizes
human Ki-67 antigen (Kosco-Vilbois et al., 1997).
Ki-Sl is another IgG mouse monoclonal antibody that was generated by immunizing
BALB/C mice with crude nuclear extracts from the human lymphoma cell line U937
(Sampson et al., 1992). This antibody recognizes a 160-kDa cell cycle–associated nuclear
antigen and can be used on sections of formalin-fixed and paraffin-embedded tissues. It is
considered useful for prognostic information in breast carcinoma (Sampson et al., 1992).
To my knowledge, the antigen recognized with Ki-S 1 is uncharacterized.
Recently, a new monoclonal antibody, MIB-5 (Immunotech, Westbrook, ME), was
generated using bacterially expressed parts of the human Ki-67 cDNA (Gerlach et al.,
1997). This antibody is equivalent to the prototype antibody Ki-67 but has the additional
advantage of being able to react with the rodent-equivalent, cell cycle–related nuclear
protein. MIB-5 antibody identifies cycling cells in embryonic and adult rat tissues fixed
with formalin and embedded in paraffin using antigen retrieval with a pressure cooker and
immunohistochernistry. The antibody is effective in both fresh and formalin-fixed tissues,
as well as in archival specimens for retrospective studies. MIB-5 antibody should also be
tried in normal and neoplastic human tissues.
Cell Proliferating Antigens 239
The aforementioned antibodies recognize different epitopes of the Ki-67 antigen with
variable survivals during fixation and embedding or differential expression during the cell
cycle. These antibodies have different affinities for the recognized epitopes and influence
immunostaining patterns (Mauri et al., 1994). It is known, for example, that although mor-
phological and cell cycle distribution of MIB-1 expression is identical to that of Ki-67 anti-
body, these two antibodies react with different epitopes of the Ki-67 antigen. Since these
antibodies are not interchangeable with one another, the cutoff values to define high- and
low-proliferating tumors that have been adopted in previous studies with Ki-67 immuno-
staining on frozen sections cannot be applied with antibodies such as MIB-1 and Ki-S5.
Tissue specimens are fixed with 4% formalin for 24 hr and embedded in paraffin.
Sections ( thick) are mounted on poly-L-lysine–coated slides, deparaffmized in
xylene, rehydrated in a descending series of ethanol, and dried for 1hr at 60°C.
Endogenous peroxidase activity is blocked with 1% in methanol for 15 min. The
slides are placed in a plastic jar filled with 0.01 M sodium citrate buffer (pH 6.0), which is
placed in a microwave oven. They are treated at 750 W for three periods of 5 min each.
During each heating cycle the buffer level is checked, and the evaporated portion is
replaced with distilled water.
The jar is removed from the oven and allowed to cool for 20 min at room tempera-
ture. Following a brief rinse in PBS (pH 7.4), 10% normal rabbit serum is applied for
20 min to block nonspecific protein immunostaining. The mouse monoclonal antibody
MIB-1 (Immunotech, Westbrook, ME) is applied overnight at 4°C in a humidified cham-
ber. For negative controls, the sections are incubated for the same duration in normal
serum in place of MIB-1 antibody. After a rinse in PBS, the sections are incubated in horse
antimouse biotinylated antibody (Vector Lab., Burlingame, CA), followed by staining with
avidin-biotin complex (Vector Elite ABC) for 1 hr. The peroxidase reaction is developed
for 1 min with 0.05% DAB (Sigma) as chromogen. The sections are counterstained with
Mayer’s hematoxylin for 1–5 min and mounted in Histomount or gelatin-glycerin.
The Ki-67 labeling index (percentage of Ki-67 positive cells) can be determined by
scoring 500–1,000 cells. The labeling index can be performed by ocular micrometry on a
Leitz or any other appropriate light microscope by using a total magnification of 400.
Immunohistochemical staining reactivity is regarded as positive when the stained cells
occupy more than 5% of the observed field. Multiple fields of a viable tumor should be
examined to minimize erroneous ratings caused by a focal or regional distribution of the
proliferating cells (Fig. 10.3). For example, renal tumors are composed of three histologi-
cal components: undifferentiated embryonic cells (blastema) and variably differentiated
epithelial and mesenchymal cells (stroma). Only nuclei with unequivocal reactivity should
be scored as positive.
Specimens from a giant-cell tumor of bone are fixed either with 10% buffered forma-
lin or 70% ethanol, decalcified with 5% EDTA in 0.1 M cacodylate buffer (pH 7.4) for 7
days and embedded in paraffin (Tsuji et al., 1997). Sections ( thick) are placed on
poly-L-lysine–coated slides (Sigma), deparaffinized, and rehydrated. Endogenous peroxi-
dase is blocked with 1 % (Sigma) in methanol for 5 min. After being rinsed with dis-
tilled water, the sections are placed in glass Coplin jars containing 10 mM sodium citrate
buffer (pH is adjusted to 6.0 with 2 N NaOH) and heated in an autoclave for 5 min at 100°C.
Before being removed from the autoclave, the jars are allowed to cool in the autoclave
until the temperature has reached 50°C. After the jars have reached a temperature of 30°C,
the sections are incubated overnight at 4°C with MIB-1 antibody at a concentration of
The sections are treated successively with biotinylated antimouse IgG anti-
body diluted 1:300 for 30 min, streptavidin-biotinylated peroxidase complex, diluted 1:50
Cell Proliferating Antigens 241
Proliferating cell nuclear antigen (PCNA) is so named because of its initial discovery
as an autoantigen found in the nuclei of proliferating cells (Miyachi et al., 1978). It was
originally detected with serum from patients with systemic lupus erythematosus, which
was found to contain an antibody against a nuclear antigen present in proliferating cells. It
was subsequently identified as an S-phase protein and named cyclin, but gradually this
term has been phased out.
Proliferating cell nuclear antigen is a 36-kDa highly evolutionary conserved eukaryotic,
acidic protein at both the protein and DNA sequence levels. Crystallographic studies have
shown that PCNA can self-associate as a trimer, forming a hexagonal ring with sixfold
pseudosymmetry and a central hole (Gulbis et al., 1996). In the center of the trimer is a
cavity that is sufficiently large to accommodate duplex DNA. This cavity is lined with
242 Chapter 10
positively charged helices facilitating interaction with the negatively charged sugar-
phosphate backbone of DNA (Cox, 1997).
The toroidal structure of PCNA has distinct front and back faces that might provide
a variety of sites for interaction with other proteins. The loop region between the twin
domains of each monomer is a highly immunogenic exposed site that is important for
interaction with other proteins. Such functional protein partners are thought to be crucial
in regulating the role of PCNA in replication and repair. The aforementioned symmetry
could define the directionality of PCNA movement as it slides along DNA.
The cell cycle is composed of S, and M phases, in addition to a resting phase
during which cells are quiescent or senescent. The expression of PCNA increases at
the end of the phase immediately preceding DNA synthesis, reaches a maximum during
the S phase, and declines through phase. It accumulates in larger subnuclear clumps in
S phase, which represents matrix-associated replication factories (Cox, 1997). This total
nuclear PCNA is tenaciously associated with the replication sites and is not removed by deter-
gents or high salt (Bravo and Macdonald-Bravo, 1987). Although PCNA is present through-
out the cell cycle, its levels are almost negligible in long-term mitotically quiescent and
senescent cells, compared with proliferating cells and increases dramatically during mitosis.
Proliferating cell nuclear antigen is involved in DNA replication as well as in DNA
repair synthesis. This antigen is required for processive DNA synthesis catalyzed by DNA
polymerase delta, which is one of the enzymes vital for DNA replication. Crystallographic
studies show that three PCNA molecules, each containing two topologically identical
domains, are tightly associated to form a closed ring (Krishna et al., 1994). The dimen-
sions and electrostatic properties of the ring suggest that PCNA encircles duplex DNA,
providing a DNA-bound platform for the attachment of the polymerase.
Accumulated evidence indicates that PCNA also plays a critical role in the initiation
of cell proliferation, and its expression is elevated almost exclusively during the S phase
of the cell cycle. However, not all studies support this observation. This antigen, as
detected by PC 10 antibody, does not accurately reflect the S-phase fraction in gastric
mucosa, as determined by bromodeoxyuridine (BrdU) labeling (Lynch et al., 1994).
Available evidence indicates that PCNA is also involved in DNA nucleotide excision-
repair. This role is exemplified by the demonstration that PCNA can be found associated
with chromatin at all phases of the cell cycle after ultraviolet irradiation in vitro (Toschi
and Bravo, 1988). Recently it was shown that not only DNA polymerase delta but DNA
polymerases beta and epsilon are also involved in the base excision repair subpathways
(Dianov et al., 1999). In addition, PCNA may be expressed by noncycling cells in vivo
which are undergoing DNA repair (Hall et al., 1993).
Immunohistochemistry
against cells that are proliferating (Mintze et al., 1995). It should be noted that markers such
as PCNA can identify those cells progressing through parts of the cell cycle but can fail to
detect significant numbers of slow or extended proliferating cell cycle populations.
Progressively longer fixation with formalin tends to reduce and eventually may
destroy antigenicity. However, in most cases, masked antigens can be unmasked by the
antigen retrieval method. Figure 10.4 (Plate 5A, B, C, D) shows the difference between
optimally fixed and overfixed tissues in the immunoreactivity of PCNA. Generally,
buffered formalin (10%) at pH 7.0 is recommended for fixation for 4hr at room tempera-
ture. Although zinc formalin was used for 4–8 hr as a fixative for studying PCNA in pig
ileum (Mintze et al., 1995), it is not recommended. PCNA retrieval on sections (
thick) of formalin-fixed and paraffin-embedded tissues can be obtained by heating in a hot
water bath at 90°C for 2 hr in 0.01 M sodium citrate buffer (pH 6.0). Alternatively, PCNA
can be retrieved by microwave heating (700 W) for two cycles of 5 min each with a 1-min
interval in tissues fixed for any length of time (see Fig. 10.3). This treatment is also effec-
tive whether tissues are fixed with formalin or Bouin’s solution. The PC10 and 19A2 anti-
bodies are preferred over MAB 424 for PCNA immunohistochemistry, and PC 10 is better
than 19A2. Table 10.2 shows recent examples of immunohistochemical localization of
PCNA antigen in various carcinomas.
The reliability of PCNA immunostaining has been questioned (Louis et al., 1991;
Harrison et al., 1993; Figge et al., 1992). In fact, some studies have ruled out a prognostic sig-
nificance for PCNA expression. The use of PCNA as a reliable marker of cell proliferation,
244 Chapter 10
for instance in intraoral squamous cell carcinoma of the head and neck, has been ques-
tioned (Nylander et al., 1997). In certain cases, PC 10 antibody–immunoreactive cells may
exceed those expected, especially in neoplasia (Hall et al., 1994).
Both the preparatory procedures and endogenous factors play a role in the unpre-
dictability of immunohistochemical results of PCNA. A number of factors—including
sample size, fixation, specific epitope involved, type and source of antibody and its con-
centration, quality of immunostaining and type of detection system, selection of micro-
scopic fields, distinguishing immunopositive nuclei from immunonegative ones, and
the threshold at which a particular staining is termed positive—are responsible for varia-
tions in the assessment results. In addition, because PCNA immunoreactivity varies
considerably within a single tumor specimen, an inexperienced observer can easily
miss the areas active in tumorigenesis, especially when the rate of proliferation is low
(Sallinen et al., 1994).
Examples of endogenous factors that may cause lack of reproducibility of PCNA
staining are given below. In some organs normal tissues show high levels of PCNA expres-
sion that is not associated with proliferation, i.e., nonproliferating cells also express PCNA
(Harrison et al., 1993; Hall et al., 1994). This phenomenon is thought to be due to changes
in PCNA regulation in association with neoplasia and the effect of growth factors on tran-
scriptional and posttranscriptional processes (Hall et al., 1990). Growth factors can mediate
PCNA expression in cells that need not enter the cell cycle. Epidermal growth factor and
TGF have been shown to increase PCNA expression in the mouse pancreas, and it has also
been demonstrated that tumors can induce PCNA expression in adjacent normal tissues
that are not proliferating (Hall et al., 1994).
The above-mentioned phenomenon may also be due to the long half-life (~15–20hr)
of PCNA. The long half-life allows cells that are no longer in the cell cycle to continue to
exhibit PCNA staining (Scott et al., 1991). Using PCNA immunohistochemistry alone it is
not always possible to make a definite distinction between actively proliferating cells and
cells arrested in the cell cycle. Thus, arrested cells become a confounding factor.
The reproducibility of PCNA immunostaining analysis can be improved by computer-
assisted image analysis (Sallinen et al., 1994). This approach also improves the repro-
ducibility of quantitation among observers. The effect of tumor heterogeneity is minimized
through this protocol because large tissue areas can be analyzed. Moreover, compared with
visual assessment, computer-assisted analysis is faster. However, even in the computerized
Cell Proliferating Antigens 245
assessment, interfield variations resulting from tumor heterogeneity are the primary reason
for the potential lack of reproducibility of the proliferation analysis. Figure 10.3 shows PCNA
heterogeneity in breast carcinoma. Considering the conflicting opinions expressed in the lit-
erature, the specificity of PCNA (and Ki-67) as a proliferation marker needs to be reassessed.
In light of the above-mentioned limitations, it seems that Ki-67 is a more specific
marker for cell proliferation than PCNA. This suggestion is strengthened by the observa-
tion that ependymal cells (which are unable to regenerate) are PCNA positive but Ki-67
negative (Funato et al., 1996). In view of the aforementioned and other factors known to
influence PC 10 labeling of PCNA, it should not be accepted uncritically as a marker of cell
proliferation in sections of paraffin-embedded tissues.
Although the following method is carried out without the typical antigen retrieval
step, it is reliable for immunohistochemical detection of PCNA using cryostat sections
(Wrobel et al., 1996). Tissues are fixed by vascular perfusion for 15 min with a mixture of
50% methanol and 10% paraformaldehyde in 10 mM phosphate buffer, followed by addi-
tional fixation by immersion for 1 hr in the same fixative. They are hydrated sequentially
in 50% and 10% methanol, washed in 0.1M phosphate buffer, and passed through a graded
series of sucrose solutions (10%, 20%, and 30%). Following immersion in Tissue TEK
OCT Compound (Miles, Elkhardt, IN), the specimens are snap-frozen in liquid nitrogen.
Cryostat sections ( thick) are mounted on gelatin/chrome-alum-coated slides
and air-dried for 30 sec. The remaining incubation steps are carried out in a moist cham-
ber. The preincubation is carried out for 45 min in the blocking buffer containing 0.1M
Tris (pH 7.4), 0.15% Thimerosal, 0.8% Triton X-100, 0.8% NaCl, 20% normal goat serum,
and 20% fetal calf serum. After rinsing three times for 10 min each in TBS consisting of
0.1 M Tris (pH 7.4), 0.8% NaCl, and 0.0015% Triton X-100, the sections are incubated
overnight at room temperature with the primary monoclonal mouse antihuman
PCNA/clone PC10 (diluted 1:3000 in PBS) (Oncogene, Uniondale, NY).
The sections are rinsed in TBS as above and incubated for 1 hr in the secondary anti-
body goat antimouse/biotinylated IgG (diluted 1:200 in the blocking buffer) (Jackson,
West Grove, PA). Following rinsing in TBS, blocking of endogenous peroxidase is accom-
plished by treating the sections with 0.002% phenylhydrazine for 10 min and with
10% for 20 min. This is followed by rinsing in TBS and incubation for 1 hr in avidin-
biotin peroxidase complex (ABC) (Vector, Burlingam, CA). The sections are rinsed in
TBS as above and developed with 0.5 mg/ml DAB in 0.1 M Tris (pH 7.4) containing
0.002% 0.04% and 0.012% They are rinsed in TBS,
dehydrated, and mounted. Controls can be carried out by omitting the primary antibody or
substituting the primary antiserum with nonimmune serum diluted 1:500 in blocking
buffer. The results of this procedure are shown in Figure 10.5.
P53 ANTIGEN
p53 antigen was discovered before the gene, but both the gene and its protein are
called p53. The term p53 was originally given to the phosphoprotein of molecular weight
246 Chapter 10
53 kDa produced by the p53 gene. This 20-kb human gene consisting of 11 exons is
located on the short arm of chromosome 17 in region 17p13.1, and its mutation occurs
most frequently in exons 5–9. The exon 5–9 region is highly conserved through evolution
and is presumably of functional importance. Approximately 95% of the reported p53
Cell Proliferating Antigens 247
mutations have been found in these exons and their intervening introns. However, p53
mutations outside exons 5–9 have also been found in human tumors (Greenblatt et al.,
1994). In many types of tumors one copy of the short arm of chromosome 17 is often lost.
Detailed studies of this chromosome have demonstrated that 17p13.1, which maps the p53
gene, is consistently lost in tumors (Baker et al., 1989).
Human p53 protein comprises 393 amino acid residues. It was first detected in SV40
transformed cells by virtue of its ability to form a stable complex with the SV40 large
T antigen (Lane and Crawford, 1979). Later it was found that many transformed cell lines,
including primary human tumor cells from patients with various types of tumors, con-
tained an elevated level of p53, whereas nontransformed cells contained only small
amounts of this protein. The genomic organization of this gene exhibits a striking degree
of similarity in different species (Furihata et al., 1995).
p53 protein contains three main functional domains: an N-terminal acidic transactiva-
tion domain, a central DNA-binding core domain, and a C-terminal homooligomerization
domain (Fig. 10.6). All three domains are required for efficient binding of p53 to recognition
sites within its physiological target genes and for transcriptional activation of these genes.
The vast majority of tumor-associated p53 missense mutations occur within the core domain.
More than 95% of the alterations in the p53 gene are point mutations that produce the
mutant p53 protein, which in most cases has lost its transactivational activity, resulting in loss
of tumor suppressor activity. p53 is the most commonly mutated gene in human cancers, and
such a gene is involved in the development of at least 50% of clinical tumors (Darnton, 1998).
Normal p53 gene is a critical controller of normal growth and homeostasis of cells
and tissues. It acts as a guardian of the genome by preventing the proliferation of cells with
248 Chapter 10
damaged nuclear DNA. This is accomplished by the production of normal (wild-type) p53
protein under normal physiological conditions. This protein is expressed at low levels and
has a short half-life due to rapid turnover mediated by ubiquitination and proteolysis.
Wild-type p53 becomes stabilized and activated in response to a number of stressful stim-
uli, including exposure of cells to DNA-damaging agents, hypoxia, nucleotide depletion,
or oncogene activation. The activation allows this protein to carry out its function as a
tumor suppressor through a number of growth-controlling endpoints. These include cell
cycle arrest, apoptosis, senescence, differentiation, and antiangiogenesis.
It is apparent from the above discussion that the two primary functions of wild-type
p53 protein are growth arrest and apoptosis. These and other functions are elicited by reg-
ulating transcription of a number of important genes. In fact, the biochemical activity of
this protein relies on its ability to bind to specific DNA sequences and to function as a tran-
scription factor. In other words, wild-type p53 protein acts on downstream genes to arrest
the cell cycle until the damaged DNA is repaired or to cause apoptosis (programmed cell
death). Apoptosis is an additional, normal mechanism for control of cellular numbers. The
concentration of wild-type p53 protein rises in cells after DNA damage, causing arrest of
the cell cycle in the (the first gap) phase and blockage of the cell cycle into the S (DNA
synthesis) phase via p21 protein. This arrest allows time for DNA repair by interaction of
wild-type p53 with downstream activators (Darnton, 1998).
As stated above, the arrest of the cell cycle is related to the activation of a number of
genes, in particular the WAF1/C1P1 gene that encodes p21 protein. The p21 impedes pro-
gression along the cell cycle at the transition, regulating cell proliferation and block-
ing DNA replication (E1-Deiry et al., 1994). This role of p21 is related to its ability to
inhibit cyclin-dependent kinases and PCNA. Therefore, cells lacking p21 may fail to arrest
the cell cycle in response to DNA damage. It can be logically assumed that lack of p21 is
an indicator of tumor aggressiveness and is correlated with p53 positivity because the
mutated p53 product is unable to activate its effector (Zlotta et al., 1999). Many cancers
show significant association between p53 abnormalities and lack of p21 expression.
However, a p21 expression independent from p53 is a common feature in some cancers,
such as malignant ovarian epithelial cell (Elbendary et al., 1996). Nevertheless, combined
immunohistological evaluation of p53 and p21 expression deserves careful consideration
in histopathological diagnosis.
p300 protein also functions in the stabilization of p53 and contributes to the p53
transactivation function in the growth arrest response to DNA damage (Yuan et al., 1999).
Accumulation of p53 is due to its stabilization rather than its increased transcription.
Deficiency of p300 results in increased degradation of p53. The N-terminal domain of p53
interacts with the C-terminal region of p300. Acetylation of the p53 C-terminal domain by
p300 stimulates the DNA binding activity of p53.
protein with a changed conformation, a longer half-life (increased stability), and disor-
dered function in terms of cellular growth. It means that mutation of this gene leads to the
loss of the guardianship of the genome, which in turn allows progression of cells with
damaged DNA through the cell cycle. Loss of control of genomic stability is central in the
development of cancer.
Both the loss of normal p53 function and the acquisition of oncogenic functions by
the mutant p53 protein can contribute to tumorigensis. Both activating and inactivating
mutations of the p53 gene can contribute to cancer progression. Inactivation of p53
protein is caused by mutations and deletions in the p53 gene or by interactions of the wild-
type p53 protein with oncogenic cellular or viral proteins, for instance, in the primary peri-
toneal carcinoma (Marchenko and Moll, 1997). Indeed, mutations in the p53 gene occur
in high frequency in most of the common types of human cancer. For example, chromo-
some 17 in more than 50% of both squamous cell and adenocarcinomas of the esophagus
harbors missense point mutations (Sasano et al., 1992). Such mutations encode altered
forms of the p53 protein. Approximately 85% of the mutations are missense mutations,
with one amino acid substituted for another and consequent alteration of p53 protein con-
formation. Thus, the oncogenic potential of p53 depends on the occurrence of a mutation
in its coding sequence.
Overexpression of p53 protein is common in human malignant tumors. Accumulation
of this protein is usually the consequence of point mutations. Immunohistochemical analy-
ses in many kinds of tumors have demonstrated a good correlation between p53 gene
mutation and overexpression of p53 protein. Such a correlation has also been detected
directly by DNA sequencing (Furihata et al., 1995). This correlation is particularly clear
for colorectal and lung carcinomas. It is well established that overexpression of p53 protein
plays an important role in the progression of cancer. However, overexpression of this pro-
tein in certain types of tumors has been reported without evidence of p53 gene mutations.
Nevertheless, nuclear staining of the majority of tumor cells accompanied by the absence
of reactivity in surrounding uninvolved tissues or stroma is the most commonly observed
pattern characteristic of the presence of a missense p53 mutation.
p73
The p53 gene product is not the only factor that induces cell cycle arrest or pro-
grammed cell death (apoptosis). Two other genes, p73 and p63, encode proteins with trans-
activation, DNA-binding, and tetramerization domains, and they share considerable
homology with p53. Like p53, these proteins also induce cell cycle arrest and apoptosis.
Each of these proteins is comprised of several isoforms. The p73 protein is a structural and
functional homologue of the p53 protein.
cAbl, a nonreceptor tyrosine kinase, regulates p73 to induce DNA damage–mediated
apoptosis. Under certain conditions such as DNA damage caused by ionizing radiation or
an alkylating agent, c-Abl is activated (White and Prives, 1999). The kinase activity of
c-Abl is induced, presumably through the action of the stress-induced ataxia telangiectasia-
mutated (ATM) gene product, a component of the DNA-damage checkpoint (Shafman et al.,
1997). The ATM protein is a widely expressed member of the protein kinases family with
similarities to phosphatidylinositol 3-kinases.
250 Chapter 10
c-Abl binds to p73 in cells, interacting through its SH3 domain with the carboxyl
terminal homooligomerization domain of p73. cAbl phosphorylates p73 on a tyrosine
residue at position 99 both in in vivo and in cells that have been exposed to ionizing radia-
tion (Yuan et al., 1999). Agami et al. (1999) have also reported that p73 is a substrate for
the cAbl kinase, and the ability of c-Abl to phosphorylate p73 is markedly increased by
As a result, p73 is able to participate in the apoptotic response to DNA damage.
The above findings define a proapoptotic signaling pathway involving p73 and c-Abl.
Unlike p53, p73 protein levels do not increase following genotoxic stress. Moreover,
although c-Abl interacts with p53 in an irradiated cell, it does not phosphorylate p53 but
still contributes to radiation-induced arrest by a p53-dependent mechanism (Yuan et al.,
1999). Although p73 is related to p53, p53 alone is the tumor suppressor. p73 protein as
yet has not been localized immunohistochemically.
Antibodies
A number of monoclonal and polyclonal antibodies to wild-type p53 and mutant p53
antigens are available and are extensively used in clinical and basic research (Table 10.3).
The binding sites for these antibodies on p53 molecule have been identified (Fig. 10.6 and
Table 10.4). These antibodies have been a major tool in the immunohistochemical detec-
tion of p53 antigen, especially in tumor tissues. The antibodies can be used for frozen or
paraffin-embedded tissues; many of them can be employed for both types of specimens,
particularly when an antigen retrieval method is used (Table 10.2). This method decreases
the immunohistochemical detection threshold of these and other antigens. Such detections
rely on the accumulation of these antigens, especially mutant p53 antigen. It should be
noted that the threshold-lowering method may detect both wild-type p53 (present in small
amounts) and mutant p53 (present in large amounts) in certain cancer-bearing tissues.
Such a possibility has been reported in the esophageal squamosa epithelium (Mandard,
Cell Proliferating Antigens 251
1998). It means that wild-type p53 may be associated with mutant p53. This hypothesis
remains to be confirmed by molecular analysis such as sequence analysis.
These antibodies are directed against different domains or epitopes of p53 protein.
The amino acid sequence of this protein is shown in Figure 10.6. These domains are dis-
tributed throughout the p53 molecule from the to the COOH-terminal end.
Most of these antibodies recognize the linear epitopes located in the amino- or carboxyl-
terminal regions of p53 protein (Legros et al., 1994a). Studies of antibodies in the sera of
mice or rabbits hyperimmunized with human p53 have confirmed that most of these anti-
bodies recognize specific epitopes located in these domains (Legros et al., 1994b). In other
words, preferential recognition of amino acid residues 1–95 and carboxyl-terminal
residues 300–393 by the antibodies exists (Schlichtholz et al., 1992, 1994). These domains
of p53 protein are highly exposed and thus readily accessible to antibodies for immuno-
histochemical detection. The central region of the protein is thought to be buried in the
interior of the molecule. However, Legros et al. (1994b) have been able to direct eight
antibodies against this region and thus define four new epitopes. To my knowledge, these
eight antibodies as yet have not been used for immunohistochemical studies.
Some of the monoclonal antibodies mentioned below recognize different p53 mole-
cule conformations. Also, detection of p53 with different antibodies depends on the time
of its synthesis. It has been suggested that the p53 epitope for antibody 1620 remains cryp-
tic immediately after synthesis in human keratinocytes and may not be exposed until late
in the life of the protein (Spandau, 1994). Furthermore, different conformations of p53
may predominate in different differentiation stages of the cell or tissue. In addition,
differentiation-specific cellular proteins and other proteins that may bind to p53 may mask
epitopes on p53 at various stages of differentiation. For example, heat shock protein 70 is
known to associate with p53 (Hainaut and Milner, 1992).
It is hoped that an understanding of the ability of various anti-p53 monoclonal anti-
bodies to recognize different conformations of p53 in cells will aid in the elucidation
of the role played by this protein in cell proliferation, cellular aging or senescence, apop-
tosis, and gene expression (repressing or stimulating). p53 has been implicated in almost
all forms of cell growth stimulation and cell growth inhibition. In addition to the informa-
tion on antibodies given below, consult Tables 10.3 and 10.4 and Figure 10.6 for their
characteristics.
252 Chapter 10
As an example, optimal dilutions of four antibodies commonly used for p53 protein
in squamous cell carcinomas are given below (Piffko et al., 1995).
CM1 1:2,000
DO-7 1:200
PAb 240 1:10
PAb 1801 1:40
Note that a wide range of dilutions of the same antibody is used, depending on the
tissue type and whether or not an antigen retrieval method is used. For example, DO-7 anti-
body has been used at a dilution of 1:10 for detecting p53 protein in esophageal carcinoma
(Yang et al., 1998), while the same antibody was employed at a dilution of 1:1,000 for
detecting this protein in breast cancer tumors (Daidone et al., 1998). There are many simi-
lar examples. Substantial differences in the quality and quantity of immunostaining of p53
are found even in the same tissue, depending on the primary antibody and the dilution used.
Immunohistochemistry
protein has a longer half-life (~12 hr), and is present in relatively large amounts, it can be
detected easily with or without antigen retrieval pretreatment (Figs. 10.7 and 10.8, respec-
tively). In comparison with formalin fixation, alcohol fixation results in increased staining
of p53 antigen, probably due to easy access of the antigen to the large antibody macro-
molecules in the absence of protein crosslinks. Allison and Best (1998) have compared the
effects of alcohol fixation with those of formalin fixation, in conjunction with microwave
heating, on the immunohistochemical demonstration of p53, PCNA, and Ki-67 antigens in
oral squamous cell carcinoma. They indicate increased nonspecific staining of p53 antigen
staining in the alcohol-fixed tissues. Similarly, fixed and treated tissues also showed p53
antigen staining in unexpected tissue components. Another adverse effect of alcohol fixa-
tion is the comparatively poor quality of cell morphology preservation, which becomes
apparent at higher magnifications. Therefore, formalin fixation is preferred.
Although a large number of immunohistochemical studies demonstrate that p53 over-
expression is positively correlated with proliferation rates in many tumor types (Table 10.5),
caution is warranted in the interpretation of such results because the presence of an hetero-
geneous population of cells within a tumor specimen is well known. This problem might
be avoided by using cell lines derived from tumors, thus obtaining a homogeneous source
of tumor cells. However, such cell lines might acquire mutations absent in the original
tumor. Moreover, part of the positive immunoreactivity could result from an accumulation
of wild-type p53 protein. Under certain circumstances, wild-type p53 protein may
256 Chapter 10
These methods may break down crosslinks between the antigen and the surrounding
proteins, allowing the epitopes to become accessible due to the mechanism(s) responsible
for the availability of epitopes for immunohistochemistry—a strategy to ensure that recog-
nition of the epitope by the antibody is required. Such a protocol is presented below.
A monoclonal antibody or its clones are directed against a specific amino acid
sequence of epitope of the antigen molecule. If such an epitope is not accessible to a given
monoclonal antibody, the interaction will not occur, resulting in false-negative immuno-
staining. To avoid this problem, multiple monoclonal antibodies against the same antigen
but reactive to different amino acid sequences can be used. This can be accomplished by
using more than one antibody simultaneously or separately. The approach almost ensures
the interaction between at least one of the antibodies and its accessible epitope, resulting
in positive immunostaining. It is also possible that more than one antibody in the cocktail
of antibodies may interact with more than one epitope. As an example, three antibodies
used in three separate studies for labeling p53 is described below.
Seven monoclonal antibodies and one polyclonal antibody used against p53 antigen
are given in Table 10.4. Each of the monoclonal antibodies shows specific affinity for a dif-
ferent range of amino acid sequences of the p53 molecule. By using three antibodies sep-
arately—DO-7 (for 21–25 amino acid sequence), Pab240 (for 213–217 amino acid
sequence), and HR 231 (for 371–380 amino acid sequence)—false-negative immunostain-
ing of p53 can be avoided (Tenaud et al., 1994). These three antibodies possess specifici-
ties for epitope distributed along the p53 molecule as shown in Figure 10.6.
The PAb 248 monoclonal antibody recognizes an epitope highly preserved between
mouse and humans (Rotter et al., 1983), which thus can be used for localizing wild-type
p53 antigen in human tissues. Using this antibody, wild-type p53 has been localized
immunohistochemically in the normal human lymphoid and epithelial cells (Pezella et al.,
1994). Sections of paraffin-embedded tissues are processed using standard antigen
retrieval with microwave heating and the immunoperoxidase technique.
Tissues are fixed with 10% neutral phosphate–buffered formalin and embedded in
paraffin, and sections ( thick) are mounted on poly-L-lysine-coated slides heated at
60°C for 30 min. The sections are deparaffinized in four changes of xylene and then rehy-
drated in a descending series of ethanol. Endogenous peroxidase activity is quenched by
immersing the sections in 1% in distilled water for 5 min. The sections are rinsed in
three changes of distilled water, transferred to a moist chamber, and covered with PBS.
The slides are placed in 0.1 M sodium citrate buffer (adjusted to pH 6.0 with NaOH)
in a plastic jar, which is transferred into a microwave oven. They are heated at 750 W for
17 min, with brief interruptions at 7 and 12 min to replace evaporated volume with distilled
water. The slides are cooled to room temperature in the citrate buffer and transferred to
PBS. Nonspecific background staining is blocked by treating the sections with diluted
258 Chapter 10
horse serum for 15 min at room temperature. After rinsing in PBS, the sections
are incubated in DO-7 antibody (diluted 1:200 in PBS) in a moist chamber for 1 hr at room
temperature. They are rinsed in PBS and treated successively with biotinylated horse anti-
mouse antiserum and avidin-biotin peroxidase complex for 30 min each. This is followed
by treating the sections with a solution of DAB (0.5 mg/ml) containing 0.009% hydrogen
peroxide. The intensity of the brown reaction product is enhanced by immersing the sec-
tions in 0.125% osmium tetroxide. The sections are lightly counterstained with hematoxylin
and successively immersed in acid alcohol, lithium carbonate solution, graded ethanol solu-
tions, and xylene. The slides are coverslipped with Permount medium. Figure 10.7 shows
the immunostaining of p53 antigen using microwave heating.
mounted on poly-L-lysine-coated slides, and immediately fixed with 100% ethanol at 4°C
for 10 min, followed by air-drying for ~45 min. The slides are stored at –70°C for 12 hr,
thawed to room temperature, and rehydrated in PBS (pH 7.4) for 5–10 min. Nonspecific
reactivity is blocked by treating the sections for 15 min at room temperature with diluted
horse serum ( Vector Mouse Elite ABC kit, Vector Labs, Burlingame, CA). This
is followed by a blocking procedure for endogenous biotin (BioGenex avidin-biotin block-
ing kit, Vector Labs) according to manufacturer’s instructions.
The sections are rinsed in PBS, followed by incubation in PAb 1801 antibody (1:100)
for 1 hr in a moist chamber. They are rinsed in PBS and treated successively with biotiny-
lated horse antimouse antiserum (30 min) and avidin-biotin-peroxidase complex (30 min)
at room temperature, using the Vector Mouse Elite ABC kit. The chromogen, a mixture of
DAB (0.5 mg/ml) and 0.009% is added. The intensity of the brown reaction product
is enhanced by immersing the sections in 0.125% solution. The sections are lightly
counterstained with hematoxylin and successively immersed in acid alcohol, lithium
carbonate solution, graded ethanol solutions, and xylene. The slides are coverslipped with
Permount medium. The results of this procedure are shown in Figure 10.9.
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Chapter 11
Estrogens
Endogenous estrogens are 18-carbon steroids and were discovered in the 1920s. They are
produced in the ovaries, adrenals, and stroma of peripheral fat. is the dom-
inant estrogen in reproductive-age women. Estrogens bind to members of the nuclear
receptor superfamily. The functions of an estrogen are mediated by specific high-affinity
estrogen receptors and located in the target cell nuclei. Estrogens clas-
sically exert their effects through the receptor mechanism of action. The estrogen enters
the cells by passive diffusion and binds to the ER. Following a series of activation steps,
the estrogen-ER complex, associated with the estrogen responsive element, functions as an
enhancer for the estrogen-responsive, element-containing genes.
Estrogen is a key intracellular modulator of the processes involved in differentiation,
development, and homeostasis. This hormone produces physiological actions within a
variety of target sites in the body and during development by activating a specific receptor
protein. Estrogen plays a crucial role in embryonic and fetal development to influence
female secondary sexual characteristics, reproductive cycle, fertility, and maintenance of
pregnancy. In addition, estrogen modulates lipid and cholesterol homeostasis in females.
The hormone also contributes to the neuroprotection seen in females after traumatic or
ischemic cerebral insults (Roof and Hall, 2000). This neuroprotection can be partly
explained by invoking estrogen’s lipid-lowering effect. Estrogen also directly affects the
blood vessel wall, microvascular vasomotor tone, and production of vasoactive substances.
Several mechanisms are responsible for these effects.
Other putative effects of estrogens include preservation of autoregulatory function, an
antioxidant effect, reduction of production and neurotoxicity, reduced excitotoxicity,
increased expression of antiapoptotic factor bcl-2, and activation of mitogen-activated pro-
tein kinase pathways. Also, there is overwhelming data indicating that estrogens enhance
survival of neurons both in vitro and in vivo (Green and Simpkins, 2000).
Estrogens are synthesized not only in females but also in males. The synthesis of this
hormone by cytochrome P450 aromatase in Leydig and Sertoli cells of the testis is well
known (Carreau et al., 1999). This cytochrome is also found in the brain, where estrogen is
important for imprinting male behavior (Beyer, 1999). There is clear evidence that the role
of ER in males is associated with the maintenance of fluid reabsorption in the head of the
epididymis (Hess et al., 1997). The loss of ER function in males interferes with the resorp-
tive function of efferent ductules, a function that is essential for fertility (Hess, 2000).
261
262 Chapter 11
The biological importance of estrogen also becomes clear considering the number of
disease states associated with altered production of this hormone or abnormalities in the
manner in which the cell responds to the biological stimulus provided to the cell by estro-
gen. It is well known that estrogen replacement therapy is associated with numerous ben-
eficial health effects, including a reduction in risk for cardiovascular disease, decreased
incidence of osteoporosis, and a significant reduction in all-cause mortality (Grady et al.,
1992; Hunt et al., 1990). Estrogen’s cardiovascular benefits result from improved profiles
as well as favorable effects on the vascular wall. It has been suggested that nuclear factor-kB
may be involved in both early and late stages of the inflammatory-proliferative process of
atherogenesis, and the negative cross-talk between ER and this factor may be a funda-
mental mechanism in estrogen’s cardioprotection. Other mechanisms are discussed by
Harnish et al. (2000).
Estrogen use is also associated with a number of clinically relevant neurological ben-
efits, including increased verbal memory, reduced incidence of Alzheimer’s disease, and
decreased neuronal damage from stroke (Sherwin and Carlson, 1997; Paganini-Hill and
Henderson, 1994; Schmidt et al., 1996). In addition, estrogen plays a positive role in
inhibiting further progress of Parkinson’s disease (Saunders-Pullman et al., 1999). There
are several possible explanations for estrogen’s effects on memory and cognition, includ-
ing modulation of neurotransmitter function and increased synaptogenesis. The direct neuro-
protective role of estrogens, as well as the proven clinical safety of these hormones,
suggest that estrogen therapy may be useful in treating neurodegenerative diseases as well
as neurotrauma such as head injury and cerebral ischemia, as mentioned above. While the
role of estrogens and their receptors in breast cancer is discussed elsewhere in this chapter,
it suffices to indicate that paradoxically, in addition to the initial promotor role of estrogens
in breast cancer, they prevent spreading of cancer cells. The protective role of against
cancer progression has also been presented elsewhere in this chapter.
ESTROGEN RECEPTORS
Three isoforms of the estrogen receptors (ER) have been identified, cloned, and char-
acterized from several species: and (Green et al., 1986; Kuiper et al., 1996;
Hawkins et al., 2000). These receptors are members of a superfamily of genes that consists
of nuclear receptors for diverse hydrophobic ligands such as steroid hormones (estrogens,
progestins, glucocorticoids, mineralocorticoids), retinoic acids (vitamin A), vitamin D,
prostaglandins, and thyroid hormones. Most members of this family are ligand-dependent
transactivators. After hormone binding and transformation, receptor-ligand complexes
interact with specific hormone response element on target genes, regulating transcription.
When not bound to the hormone, ERs exist in an unactivated, untransformed state
(as a monomer) and complex with heat shock proteins. In the estrogen-binding state, the
receptors undergo physico-chemical changes, including phosphorylation at specific serine
and tyrosine residues that are accompanied by conformational changes (Arnold et al.,
1997). These changes result in the dissociation of heat shock proteins from the activated
complex and formation of a 5S homodimer with high affinity for estradiol and DNA. The
transformed dimer binds to its specific estrogen response element located in the promoter
region of estrogen-responsive genes, regulating their transcriptional activity. Estrogen
Estrogens 263
Previously only a single type of ER was known to exist as the mediator of the
genomic effects of estrogen in specific target tissues. Later, cloning of a gene encoding a
second type of estrogen receptor was reported in the mouse, rat, and humans
(Kuiper et al., 1996; Vladusic et al., 1998). To distinguish between these two ERs, the ini-
tial receptor is termed This development has prompted a reevaluation of the estrogen
signaling system.
In mammals, the gene is mapped to the q22-24 band of chromosome 14, while
the gene is mapped to the long arm of chromosome 6 (Enmark and Gustafsson,
Estrogens 265
1999). In addition, several isoforms of these two subtypes were recently reported. These
are transcribed either by alternative exon splicing or usage of different promoters of a sin-
gle gene (Friend et al., 1995; Chu and Fuller, 1997; Ogawa et al., 1998). Estrogen recep-
tor gamma is derived through gene duplication. The existence of multiple forms of the
receptor may explain the pleiotropic actions of estrogens in diverse tissues or species.
The discussion in this volume pertains to receptors and and
Estrogen receptor alpha and consist of a hypervariable N-terminal domain that
contributes to the transactivation function (A/B), a highly conserved central domain respon-
sible for specific DNA binding, dimerization, and nuclear localization (C), an estrogen-
binding domain (E), a hinge region domain (D), and a domain (F) whose function is not
known (Tonetti and Jordan, 1997).
A high level of homology exists between and especially in the DNA-binding
and estrogen-binding domains. These two ERs can form homodimers with themselves or
heterodimers, providing three potential pathways for estrogen signaling. However,
and differ in the C-terminal ligand–binding domain and in the N-terminal transacti-
vation domain. The difference between ER subtypes in relative ligand binding affinity and
tissue distribution explains the selective action of ER agonists and antagonists.
The human is a complex genomic unit exhibiting alternative splicing and pro-
moter usage in a tissue-specific manner. This observation demonstrates the importance of
transcriptional control in the regulation of expression. However, the mRNA sta-
bility is subject to hormonal control, suggesting that the regulation of the expression of this
receptor may also occur at a posttranscriptional level (Saceda et al., 1989). Note that
human mRNA has a relatively short half-life of approximately 5 hr in the breast car-
cinoma cell line MCF-7 after actinomycin D treatment; actinomycin D is the transcrip-
tional inhibitor (Kenealy et al., 2000).
Six functional regions (A–F) are recognized in the molecule, which show dif-
ferent degrees of amino acid sequence conservation. Human is comprised of 595
amino acids with a molecular weight of 66–70 kDa. Conserved domain organization
responsible for specific functions of is DNA binding, ligand binding, dimerization,
protein binding, and transcriptional activation. The hypervariable A/B domain in the
amino-terminal region of exhibits little or no conservation between species. This
region contains an activation function, is important for transactivation, and is responsible
for gene and cell specificity.
Region C corresponds to the DNA binding domain and is responsible for specific bind-
ing of the receptor to estrogen response elements located in target genes. Region D is the
hinge region, which separates the DNA-binding domain from the ligand-binding domain.
This region also facilitates conformational changes in the receptor molecule during activa-
tion and is important in receptor dimerization. Region D and the C-terminal portion of region
C contain nuclear localization signals and are responsible for nuclear localization. Region E
is located in the C-terminal portion of the receptor and is responsible for ligand binding. This
region contains a second activation function domain, involved in transactivation in conjunc-
tion with A/B domain. The exact functional role of region E is not clear, although it may play
266 Chapter 11
a role in distinguishing between agonist and antagonist binding to the receptor molecule
(Montano et al., 1995).
is predominantly expressed in specific tissues, such as breast, uterus, and vagina.
The receptor plays a key role in many normal physiological processes, ranging from
female sexual development and reproduction to liver, fat, and bone cell metabolism. It is
also involved in the biology of breast cancer and is used clinically as an important prog-
nostic factor (Fig. 11.3). Significant amounts of the have been detected in more than
60% of human breast cancers. Approximately 70% of the tumors respond to
antiestrogen therapy compared with only ~5% of the tumors.
In spite of the usefulness of ER immunohistochemistry in the diagnosis and prognosis
of the breast cancer, the published data are not always in agreement. The discordant
immunohistochemical ER results reported in the literature are partly owing to the use of dif-
ferent monoclonal antibodies. Generally, different antibodies recognize a specific epitope
within the domains over the entire length of the ER. For example, the difference between
the reactivity of ERID5 monoclonal antibody (which targets an epitope in the A/B region)
and H222 monoclonal antibody (which targets an epitope in the E region) observed in
breast tumors is considered to be due to the presence of ER variants (Elias et al., 1995).
Also, by developing monoclonal antibodies to specific domains of ER, the presence of
structurally defective ER in breast tumor has been demonstrated (Traish et al., 1995). Thus,
Estrogens 267
Estrogen receptor beta has been cloned from rats, humans, and several other
species (Kuiper et al., 1996; Mosselman et al., 1996; Lakaye et al., 1998). Human is
expressed in multiple isoforms with various amino acid numbers. Recent studies document
that the expressed full-length human is comprised of 530 amino acids (Fuqua et al.,
1999). The DNA-binding domains of human and human are highly homolo-
gous, approaching 96%, while the ligand-binding domain shows only 59% homology.
The N-terminal A/B domain, hinge region, and F domain are distinct in sequence between
and The binds the natural hormone (estradiol) with affinity similar to
268 Chapter 11
that of However, it should be noted that the differences in the distribution and
structure between these two receptors suggest that the two isoforms have different biological
activity.
The is present in the nucleus of a wide range of normal adult human and rat
tissues, including breast, ovary, fallopian tube, uterus, lung, kidney, brain, heart, prostate,
testis, oviduct, adrenal, seminal vesicle, and bladder (Saunders et al., 1997; Taylor and
Al-Azzawi, 2000). The presence of and in the normal human breast tissue is
shown in Figure 11.2. Specifically, this receptor is located in the epithelial cells in most
male tissues, including the prostate, the urothelium and muscle layers of the bladder, and
the Sertoli cells in the testis. In the uterus, both and are present in epithelial cells
lining the lumen and glands. In the lung, is found in the cells lining the bronchioles
and alveoli and smooth muscle.
It has been known for some time that some genomic actions of estrogen cannot be
attributed to either or For example, continues to protect against
vascular injury in both and knockout mice (Karas et al., 1999). This evidence
suggests the presence of additional types of estrogen receptors. Recently, the presence of
a third type of estrogen receptor, in a teleost fish, the Atlantic croaker (Micropogonias
undulatus), was reported (Hawkins et al., 2000). This receptor is thought to have arisen
through gene duplication from early in the teleost lineage. Receptors and are
also present in this vertebrate species. The three ER subtype receptors are genetically dis-
tinct and have different distribution patterns in this vertebrate. These three subtypes of
receptors have distinct functions, at least in the hypothalamus.
is more widely distributed than and when both receptors are present in a
tissue, the former is predominant. is present in the nucleus of a wide range of normal
adult human and rat tissues, including breast, ovary, oviduct, fallopian tube, uterus, prostate,
testis, seminal vesicle, bladder, and lung (Saunders et al., 1997; Taylor and Al-Azzawi,
2000). Based on mRNA analyses, this receptor is expressed in the central nervous system,
cardiovascular system, immune system, and gastrointestinal tract (Gustafsson, 1999). In the
lung, is found in cells lining the bronchioles and alveoli and smooth muscle. Moderate
to high expression of is found in uterus, testis, pituitary, ovary, kidney, epididymis, and
adrenal gland. In the uterus, and are expressed in epithelial cells lining the lumen
and glands.
Although distribution is closely related to the expression of in some
tissues, the expression of these two receptors does not seem to be linked. Some
cells lack while other cells show both of these receptors, and still other cell
types are and A few examples follow. In the endometrium,
both and are present in luminal epithelial cells and the nuclei of stroma cells,
while expression is weak or absent in the endometrial glandular epithelia. In the
Estrogens 269
ovary, is present abundantly in multiple cell types, such as granulosa cells in small,
medium, and large follicles, theca and corpora lutea, whereas is undetectable in these
cell types (Saunders et al., 1997). Breast and pituitary contain both receptors, whereas
prostate shows positive immunoreactivity for but negative for An extensive list
of the immunohistochemical distribution of and in adult human tissues is pre-
sented by Taylor and Al-Azzawi (2000).
The ER and the progesterone receptor (PR) belong to the steroid hormone receptor
family of ligand-inducible transcription factors, which play a key role in the development
and progression of breast cancer. Although breast is influenced by many hormones and
growth factors, estrogens play an important role in promoting the proliferation of both
normal and neoplastic breast epithelium. The influence of estrogens on the proliferative
activity of mammary epithelial cells is mediated by at least three mechanisms: receptor
mediation, autocrine/paracrine loop, and negative feedback (Kumar et al., 1987; Huff et al.,
1988; Soto and Sonnenschein, 1987). However, these mechanisms have not been precisely
defined as to their role in the normal development and differentiation of the breast or in
the initiation and progression of the neoplastic process. Because normal epithelium con-
tains receptors for estrogen and progesterone, the receptor-mediated mechanism is a major
player in the hormonal regulation of breast development.
Considerable amounts of ER are present in more than 50% of primary human breast
cancers. The presence of ER in primary tumors identifies patients with a lower risk of
relapse and better overall likelihood of survival. Moreover, response to endocrine therapy
mostly depends upon the presence of ER and PR, the latter indicating functional ER sig-
nal transduction because PR expression is regulated by ER. Consequently, ER determina-
tion has become an established procedure in the management of patients with breast
cancer. Approximately 50–70% of patients with recurrent disease who had ER-positive
primary tumors respond to hormonal treatment compared with only ~5% of patients with
ER-negative tumors, suggesting a strong correlation between the growth of breast tumors
in vivo and the presence of ER. However, the duration of response is limited because of
progression to an estrogen-independent state of the tumor. In other words, patients with
ER-positive breast cancer have a more favorable clinical course and prognosis and longer
disease-free intervals than those with ER-negative cancer. Therefore, determination of the
ER content of breast cancer tissue is indispensable for selecting a regimen of treatment
when there is a relapse or for predicting the prognosis.
Some prognostic factors predicting failure of endocrine therapy are known. The pres-
ence of epidermal growth factor (EGF) indicates poor prognosis and is correlated with lack
of response to endocrine therapy in recurrent breast cancer. It is recognized that expression
of EGF receptor is inversely related to ER expression in malignant breast tumors and
breast cancer cell lines, both at the protein and mRNA levels (Klijn et al., 1992). However,
~50% of ER-positive tumors contain EGF receptors. But ER and EGF receptors are rarely
expressed simultaneously in the same malignant cell. The likely reason is that both receptor
signal pathways become uncoupled during malignant progression. This and other evidence
documents the heterogeneous nature of primary breast tumors.
270 Chapter 11
A double immunohistochemical method has been used for determining the expression
patterns of ER, PR, and EGF receptors in breast biopsies (Van Agthoven et al., 1994). It
was demonstrated that ER/PR and EGF receptors in breast tumor cells were inversely
related at the single cell level. However, the expression of these three receptors in individual
normal luminal cells was not mutually exclusive.
It is well known that the ER level is important as a prognostic and predictive marker
in breast cancer patients. ER status is correlated with endocrine therapy. Tamoxifen is the
most common partial antiestrogen and is used for treating all stages of breast cancer.
Clinical trials demonstrate that tamoxifen is useful for the treatment of breast cancer
(Fisher et al., 1998). It acts by competitively binding to ER, but its activity ranges from
full estrogen antagonist to a partial agonist in different tissues. Its effectiveness varies with
the prevailing estrogenic environment (Furr and Jordan, 1984).
The effectiveness of tamoxifen can be evaluated in relation to Ki-67 antigen (a nuclear
proliferation marker), which is useful in determining the prognosis of breast cancer. (Ki-67
antigen is discussed in Chapter 9.) The relationship between ER levels and Ki-67 antigen
expression before and after tamoxifen treatment has been investigated (Dardes et al., 2000).
Immunohistochemical studies demonstrate a decreased Ki-67 labeling index after tamox-
ifen treatment in ER-positive patients. Patients with down-regulation of ER expression also
show decreased Ki-67 labeling index after tamoxifen therapy. This phenomenon may be
based on the ability of tamoxifen to induce apoptosis and reduce the levels of ER as a tran-
scription factor (Dardes et al., 2000) This and other studies indicate that short-term
(4 weeks) tamoxifen therapy decreases the proliferation of breast cancer in ER-positive
breast tumor specimens. In relation to ER level there is no difference in the Ki-67 labeling
index level between pre- and posttamoxifen treatment of ER-negative patients.
Recent studies suggest that postmenopausal patients older than 50 with ER-negative
breast cancer, who do not respond well to either hormonal therapy with tamoxifen or adju-
vant chemotherapy, may have a significant response to vaccination with autologous tumor-
associated antigens (Jiang et al., 1999). Such a vaccination results in a reduction in serum
IL-6 concentration in patients with ER-negative breast cancers; it is known that estrogen
represses IL-6 expression. This approach does not have a direct cytotoxic effect on cancer
cells but is an attempt to promote mechanisms of rejection of the tumor by the host.
ANTIBODIES
Estrogen receptor comprises several structural domains with specific and overlapping
functions. A number of monoclonal and polyclonal antibodies are available, some of which
show affinity for specific domains of the ER molecule. The antibodies discussed below are
efficient tools for ER immunohistochemistry on sections of formalin-fixed, paraffin-
embedded tissues and facilitate the cellular site expression of and receptors in
human and rat tissues. Most of these antibodies are used for labeling ERs in breast tissue
in conjunction with pretreatment with antigen retrieval methods.
Estrogens 271
of breast cancer than does H222. According to Goulding et al. (1995), however, using the
Spearman’s rank correlation method, a highly significant correlation is found between the
H scores using antibody ERID5 or antibody H222. For manual immunohistochemistry
with antibody H222, the sections are stained using Abbott’s ER-ICA monoclonal antibody
kit. H222 was the first antibody applied to paraffin sections.
Another monoclonal antibody, NCL-ER-6F11, has been generated by using a recom-
binant ER protein instead of the peptide antigen approach (Bevitt et al., 1997). Because
multiple epitopes are presented by the recombinant protein at immunization, production
of a greater number of hybridomas is expected. NCL-ER-6F11 antibody is specific to
human (see Fig. 11.3) and does not bind to recombinant human NCL-ER-6F11
antibody compares favorably with ERID5 antibody, which is also generated using
recombinant ER.
Mouse monoclonal antibody AER311 (Neomarkers, Fremont, CA), unlike ERID5,
recognizes the carboxyl-terminal domain and reacts only with wild-type estrogen, except
for DNA-binding truncated protein (Huang et al., 1996). Immunohistochemistry using
ERID5 or AER311 can distinguish hormone-binding truncated protein from wild-type
estrogen. The estrogen-enzyme immunoassay method recognizes various mutant proteins
that can also be detected only by the ERID5 antibody and not by the AER311 antibody
because these two antibodies recognize different targets on the ER molecule. As stated
above, the AER311 antibody does not react with ERs that lack the hormone-binding
domain. Immunohistochemical studies using these two antibodies have shown that a num-
ber of palpable breast cancers lack the carboxyl terminal in the ER, regardless of wild-type
ER mRNA expression (Hori et al., 1999). Other monoclonal antibodies include D75P3,
CC4-5 (Ventana Medical Systems) and 6F11 (Vector Laboratories, Burlingame, CA).
Compared with CC4-5, 6F11 gives more intense nuclear staining and less cytoplasmic
reactivity.
Saunders et al. (1997) have raised a polyclonal antiserum using a peptide specific for
The peptide (CLSKAKRNGGHAPRVLEL) corresponding to amino acids 196-213
of rat was conjugated to keyhole limpet hemocyanin and used to immunize rabbits
according to standard procedures. Polyclonal IgGs were purified from serum on a Hitrap
protein A Sepharose column based on the manufacturer’s instruction (Pharmacia).
The monoclonal mouse antibovine (05-394) antibodies directed against SDS-
solubilized calf uterus and polyclonal rabbit antirat (06–629) antibodies devel-
oped against the N-terminal region of the human sequence are commercially
available (Upstate Biotechnology, Lake Placid, NY). Another polyclonal rabbit antirat
(310) antiserum developed against the C-terminal region of the human sequence
is also commercially available (Affinity Bioreagents Inc., Golden, CO).
To test possible interactions between various ER functional domains, monoclonal
antibodies to various regions of the ER have been developed (Traish and Pavao, 1996).
Monoclonal antibody F9 was developed against a synthetic 30-mer hybrid oligopeptide.
Another monoclonal antibody, NMT-1, was raised against 15-mer peptide from the N-
terminal A/B region (amino acids 240–154). Monoclonal antibody 213 was generated
against peptide AT3 in the DNA-binding domain (amino acids 247–263).
The effects of binding these site-directed, monoclonal antibodies to specific regions
of the ER molecule on the conformation of this molecule have been determined. Such
studies indicate that the conformational change within a small stretch of the ER molecule
Estrogens 273
caused by the binding of an antibody is transmitted to another distal region of the recep-
tor. This phenomenon is exemplified by the binding of the antibody NMT-1 to the A/B
region of the receptor, which causes the release of the antibody from its epitope in the
DNA-binding region. Thus, the binding of these site-directed, monoclonal antibodies to
specific regions of the ER molecule affects the conformation of this molecule.
IMMUNOHISTOCHEMISTRY
receptors, ER and PR (PgR) (Barnes and Millis, 1995). Note that although ER or PR
nuclear expression alone predicts response to hormonal therapy, combined ER/PR pheno-
types have more precise predictive ability than either factor alone. Prognostic and predictive
factors in breast cancer commonly assessed by immunohistochemistry are reviewed by
Mohsin and Allred (1999). Recently, Taylor and Al-Azzawi (2000) have carried out
an extensive immunohistochemical investigation identifying human tissues that express
receptor and comparing the expression pattern of with that of
The detection of immunohistochemical staining semiquantitatively is preferred over
other methods because by using specific, monoclonal antibodies the ER positive cells are
identified by a distinct nuclear labeling. The staining can range from weak to intense,
depending upon the number of recognizable ERs present in the individual nuclei. In fact,
some evidence indicates that computerized quantitation is no better than semiquantitative
visual analysis (Schultz et al., 1992; Remmele and Schicketanz, 1993). Also a strong cor-
relation has been found between results obtained with true-color, computer-assisted image
analysis and semiquantitative scoring (Kohlberger et al., 1999).
However, other studies indicate that computerized image analysis is superior to semi-
quantitative assessment because of higher accuracy and reproducibility (McClelland et al.,
1991). Quantitative immunostaining analysis of ER using the Cell Image Analysis System
SAMBA 4000 has been carried out (Esteban et al., 1994 a, b, c; see also page 105). This
system optimizes measurements, while human deficiencies are reduced to a minimum.
Moreover, semiquantitative scoring requires more experience, know-how, and training.
Also, ER immunohistochemistry unfortunately has not been subjected to rigorous statisti-
cal analysis to define cutoff values associated with clinically meaningful endpoints such as
response to endocrine therapy. Automated electronic analysis in the near future will estab-
lish reliable observer-independent evaluation of immunohistochemical variables. Although
some of the computer-assisted image analysis equipment is too expensive for daily, rou-
tine use, it is possible to analyze ER and PR expression routinely and inexpensively with
good correlation to clinical outcomes using a relatively inexpensive standard IBM PC and
Adobe Photoshop software (Lehr et al., 1997). A similar type of analysis has been carried
out on endometrial samples from patients treated with hormone replacement therapy to
help predict clinical outcomes (Wahab et al., 1999).
A number of variations of the immunohistochemical methodology are available to
localize ERs. The expression of these receptors can be studied on paraffin sections
or frozen sections, with or without antigen retrieval application, although the latter appli-
cation is preferred. Various heating treatments, such as microwave heating, autoclave heat-
ing, and boiling on a hot plate, are equally effective in unmasking ERs in paraffin sections.
An example of the distribution of ERs in the pituitary gland is given below.
Using immunohistochemistry in conjunction with autoclave antigen retrieval, cellular
distribution of and has been accomplished on paraffin sections of fetal and adult
rat pituitary glands (Nishihara et al., 2000). The expression of in the fetal pituitary is
lower than that during the adult period and is limited to the nuclei of anterior lobe cells
from day 17 of gestation. In contrast, is present in the nucleus as well as in the cyto-
plasm in both the anterior and posterior lobes during the fetal period from day 12 of ges-
tation. The distribution of in the adult pituitary is mainly restricted to the anterior
lobe. It seems that plays different roles in the pituitary during the fetal and adult peri-
ods. The above evidence also indicates that oncogenetic changes in the expression of these
Estrogens 275
receptors are not uncommon. Note that the quantitation of prognostic markers, including
ERs, is hampered by a time-related loss of antigenicity, especially in paraffin sections on
glass slides (see page 84).
breast cancer tumors and cytological preparations (Allred et al., 1990; Masood, 1989;
Shimada et al., 1985). However, immunohistochemistry of paraffin sections is both less
sensitive and reproducible compared with frozen-section immunostaining. One of the dis-
advantages of frozen sections is poor preservation of cell morphology. For this and other
reasons, the use of paraffin sections has become more popular than frozen sections.
Note that controversies over the technical and clinical validation of immunohisto-
chemistry have not been completely resolved. Whether or not this method should com-
pletely replace biochemical ligand-binding assays remains controversial. Despite this
cautionary statement, it is true that the specificity of immunohistochemistry is theoretically
valid because it is based on the use of well-characterized monoclonal antibodies raised
against epitopes restricted to the ERs.
The dextran-coated charcoal (DCC) assay measures the hormone-binding capacity of the
cytosol fraction of the tumor tissue (Lee and Chan, 1994). Fresh tissue (more than 100 mg) is
immediately frozen, homogenized, and the cytosol fraction is extracted. Aliquots of the
cytosol are incubated with radiolabeled estradiol with and without 100-fold molar
excess of nonradiolabeled competitor (diethylstilbestrol) to displace radiolabeled steroid
from the low-capacity specific binding sites (ER), but not from the high-capacity nonspecific
binding sites in the cytosol. The unbound steroid is removed by selective adsorption on DCC.
The receptor binding capacity, reported in femtomoles of radiolabeled steroid bound per
milligram of cytosol protein, is calculated by subtracting the level of nonspecific cytosol-
bound radioligand (radiolabeled steroid with excess of competitor) from the total
cytosol-bound radioligand (radiolabeled steroid without competitor).
Two methods are available to determine the estrogen (and progesterone) status in the
tissue samples: biochemical assays (e.g., dextran-coated charcoal [DCC] method) and
immunohistochemistry. Immunohistochemistry is very useful in estimating prognosis and
monitoring therapy in breast cancer. An advantage of immunohistochemistry over the
DCC assay is that the former can be used with small tissue samples, and it also facilitates
morphological evaluation of individual tumor cells. In contrast, because ligand-binding
assays only detect estrogen receptors in an unoccupied form, they are prone to interference
by other endogenous hormones and may give rise to unreliable results. Furthermore, such
assays preclude assessment of estrogen receptor contents of individual cells. Scoring can
be performed according to the two different semiquantitative methods of Remmele and
Stegner (1986) and Reiner et al. (1987). Specific staining is identified by distinct colored
staining of nuclei with the histoscore system, which considers both intensity of staining
and percentage of stained cells.
According to the method of Remmele and Stegner (1986), intensity is graded from
0–3, where 0 = no staining, 1 = weak staining, 2 = moderate staining, and 3 = strong
staining. The percentage of stained cells is categorized as follows: 0 = 0%, 1 = 1–10% ,
Estrogens 277
2 = 11–33%, 3 = 34–55%, and 4 = 67-100%. The two values obtained from the staining
intensities and percentage of positive cells are multiplied. This method results in values of
0 (negative) and 1, 2, 3, 4, 6, 8, 9, or 12 (positive).
On the other hand, according to the second system (Reiner et al., 1987), the score is
acquired by adding the two values (staining intensities and percentage of positive cells)
and obtaining the values 0 (negative), 2 and 3 (low positive), 4 and 5 (intermediate posi-
tive), or 6 and 7 (high positive). In contrast to the complex Remmele-score, the Reiner
score is relatively simple and easily applicable, making it more advantageous in daily rou-
tine investigations (Biesterfeld et al., 1996). Moreover, the Reiner score has better repro-
ducibility than the Remmele score. It is emphasized that at least three different fields on a
slide are observed, and 100 cells are counted in each field. More than one observer should
participate independently in the scoring in a blind test. Following scoring, a consensus
score should be established among the observers.
Note that immunohistochemical reactions do not always yield homogeneous results
but are subject to variations from slide to slide and from case to case. Because tumors are
heterogeneous, caution is warranted in assuming that a particular section of the tumor is
representative of the whole tumor (see page 14 for discussion on tumor heterogeneity).
(Dako) and heated in a microwave oven as described above. After incubation in normal
swine serum (Dako) for 30 min, the sections are incubated in the 65-kDa antirat estrogen
antibodies, diluted 1:100 with PBS for 12hr. Detection is achieved as described
above, except that the secondary biotinylated rabbit antimouse immunoglobulin is
replaced with the biotinylated swine antirabbit antibody (Dako).
To localize in frozen sections, antigen retrieval with heating is not required.
210-180-C050 antibodies (Alexis Corporation, Nottingham, UK) are obtained by immu-
nizing rabbits with synthetic peptides representing the C-terminal amino acid residues
467–485 of human estrogen.
by Tris-saline buffer (TSB). Immunostaining is performed using the Dako K1900 ER/PR
staining kit (Dako Corporation, Carpinteria, CA). It includes all ready-to-use reagents,
antibodies, and detection system. The smears are covered with hydrogen peroxide for
5 min, then with distilled water, and then placed in TSB for 5 min. They are incubated in
primary monoclonal antibodies (Dako K1900) for 30 min and then washed with TSB for
5 min. This is followed by incubation with biotinylated linking antibody for 10 min and
washing in TSB for 5 min. The smears are incubated with strepavidin-peroxidase for
10 min and then washed with TSB for 5 min. They are treated with chromogen substrate
DAB for 10 min and rinsed with tap water for 5 min. Counterstaining is accomplished with
hematoxylin for 20 sec. The smears are dehydrated, cleared and mounted. The results of
this procedure are shown in Figure 11.6.
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Chapter 12
The HER-2/neu oncogene plays a key role in breast and many other cancers. Methods,
including immunohistochemistry, are available to analyze tumor HER-2 status (Fig. 12.1).
Elucidation of the human genome is expected to significantly improve many aspects of
health care, especially the diagnosis and management of cancer. Genetic discoveries are
already resulting in the development of specific, effective, and less toxic cancer drugs than
many of those in current use. An important example of a less toxic reagent is Herceptin,
which targets HER-2/neu gene product, p185 (HER-2).
HER-2/NEU ONCOGENE
281
282 Chapter 12
colorectal adenocarcinomas (Koeppen et al., 2001), skin cancer (Krähn et al., 2001), and
lung cancer (Cox et al., 2001).
The amplification of the HER-2/neu oncogene is thought to have significance in the
early stages of malignant transformation, especially in the breast and ovary, and thus may be
considered a prognostic marker of these carcinomas. A statistically significant correlation
between the amplification of this gene and survival of patients with breast and ovarian
tumors has been reported (Slamon et al., 1989; Carr et al., 2000). Because the gene is over-
expressed in breast cancer patients, determination of its prognostic significance is being
evaluated. This oncogene is indeed an independent prognostic indicator of a subset of breast
cancers that are at high risk of early recurrence, regardless of tumor grade, estrogen/prog-
esterone receptor status, or lymph node status (Carr et al., 2000). However, according to
Slamon et al. (1987), association of HER-2/neu amplification and poor prognosis is
stronger for patients with lymph node metastases than without lymph node involvement.
The HER-2/neu gene is overexpressed in 25–30% of human breast cancers, and in
~90–95% of these cases, the overexpression is a direct result of gene amplification. The
amplification or overexpression of this gene is associated with shorter disease-free survival
as well as overall survival. Appropriate follow-up studies have confirmed the prognostic
association between the HER-2/neu gene alteration and clinical outcome for both node-
negative and node-positive breast cancers (Press et al., 1997)
The HER-2/neu gene is also overexpressed in epithelial ovarian cancer. Several studies
have reported that overexpression of this gene is associated with poor survival rates in
Oncoprotein 283
advanced epithelial ovarian cancer (Berchuck et al., 1990; Bast et al., 1992). Anreder et al.
(1999) have also reported poor survival rates for patients showing overexpression of the
oncogene in the ovarian surface epithelial tumors.
Note, however, that some types of primary tumors, such as breast cancer, may contain
elevated p185 protein or HER-2 mRNA levels in the absence of detectable gene amplifica-
tion, suggesting that in some cases gene amplification is not strictly required for p185
expression. In other words, the presence of this protein in some cancer cells itself is an index
of tumor aggressiveness, regardless of HER-2/neu amplification (Dati et al, 1990).
It should be noted that although HER-2/neu gene is considered to be the target gene
for amplification at chromosome bands 17ql2–q21, the amplicon harbors several closely
located genes such as retinoic acid MLNs 50, 51, 62, and 64, gastrin,
hormone and topoisomerase (Järvinen et al., 1999). Many
breast tumors with HER-2/neu amplification show simultaneous amplification or deletion
of topoisomerase gene. Amplification of the HER-2/neu is followed by complex sec-
ondary genetic aberrations, which lead to amplification or deletion of the topoisomerase
gene in a majority of tumors. A simple molecular mechanism suggested for this
phenomenon is that the amplification of the chromosomal segment includes both genes. The
importance of this information is that the gene copy number aberrations of topoisomerase
may divide HER-2-amplified breast tumors into clinically meaningful entities.
HER-2 ONCOPROTEIN
signaling may be sufficient to facilitate tumor cell transmigration and metastasis. The
molecular mechanism(s) of HER-2-induced endothelial cell retraction is unknown.
It is interesting to note that expression and secretion of an aberrant HER-2 splice
variant has been reported in various cell lines and tissues, which can interfere with the
oncogenic HER-2 activity (Aigner et al., 2001). Expression of this truncated 100-kDa
HER-2 variant encodes the extracellular domain of HER-2 and inhibits growth
factor–mediated tumor cell proliferation. The exact role played by this variant during the
progression of human cancer is not clear.
HER-2 OVEREXPRESSION
Overexpression of the HER-2 protein is one of the most studied molecular changes
that occur in human cancer. The main cause of HER-2 overexpression is HER-2/neu gene
amplification, although protein overexpression has been observed in some subsets of breast
carcinoma despite a lack of gene amplification (Persons et al., 1997). It is reasonable to
suggest that HER-2 may not have an identical role in all tumors. It is known that HER-2
overexpression is able to transform cells in vitro, although catalytic activation of this recep-
tor may also be involved in other functions. In fact, the precise functional significance of
this phenotype is uncertain. This uncertainty is reinforced by evidence that HER-2 overex-
pression shows stronger association with preinvasive rather than with advanced disease.
Moreover, extensive apoptosis occurs in preinvasive diseases. This and other evidence
raises doubts about the precise significance of the HER-2 phenotype in vivo.
Published immunohistochemical studies of overexpression of HER-2 report a wide
range of overexpression rates, ranging from 9 to 60%. The likely reason for this variation is
the use of different antibodies, tissue types, and fixation and staining protocols. In addition,
different scoring systems result in interpretation differences. To minimize these variations
the use of a uniform immunohistochemical method, such as the HercepTest, is recom-
mended. The HercepTest kit provides standardized procedure and evaluation criteria.
Among molecular markers for sporadic breast cancer, p53 protein and HER-2 recep-
tor protein have retained special attention. Genetic alterations resulting in overexpression
of these two proteins are a frequent finding in early stages of invasive ductal carcinomas
of the breast, suggesting a role in breast cancer pathogenesis and possibly a function in
disease progression.
The detrimental consequence of simultaneous expression of these two proteins has
been found even in node-negative mammary carcinomas (Albanell et al., 1996). Moreover,
HER-2 and p53 overexpression individually correlates with a higher growth rate in mam-
mary carcinomas. More recently, Rudolph et al. (2001) also provided evidence that over-
expression of either HER-2 or p53 resulted in an increased cycling ratio, which is thought
to be additive in tumors overexpressing both proteins. p53 expression is significantly more
prevalent in young patients, whereas HER-2 expression shows only a trend toward a higher
occurrence in the young.
Oncoprotein 285
Astrocytic Tumors
It is difficult to distinguish between benign and malignant astrocytic tumors. Some low-
grade astrocytomas behave similarly to anaplastic ones. This problem may be solved through
determining the proliferative index of antigens such as HER-2, p53, PCNA, p21, EGF, and
EGFR. Such an immunohistochemical study has been recently carried out by Bian et al.
(2000). This study indicates that aberrations of HER-2, p21, EGF, and EGFR might be early
events in the initiation and progression of astrocytomas, but they are unrelated to histologi-
cal grade and prognosis of astrocytomas. On the other hand, p53 overexpression is involved
in all the stages, while PCNA might be important in evaluating astrocytoma malignancy.
In this study, sections were pretreated with 0.1% trypsin containing 0.1% prior to
treatment with 0.3% in methanol to block endogenous peroxidase.
Bladder Carcinoma
Wood et al. (1991) have used the Southern hybridization method for detecting DNA
amplification and a possible structural rearrangement of the HER-2/neu oncogene in 1 of 12
bladder tumors. Amplification of this oncogene in the tumor was sixfold that of oncogene
found in placental DNA. Approximately 36% of the tumors studied overexpressed HER-2
mRNA, which was 3- to 38-fold that of normal urothelium. HER-2 overexpression occurred
in superficial and invasive tumors. Deoxyribonucleic acid amplification occurs infrequently
in bladder carcinoma, in contrast to its occurrence in some other carcinomas.
Immunohistochemical analysis has shown that p185 HER-2 polyclonal antibody is specific
for HER-2 protein overexpression in bladder carcinoma. This study was carried out prior to
the use of Herceptin.
Ewing's Sarcoma
Ewing’s sarcoma is the second most common malignant bone tumor in children. The
majority of patients have microscopic metastases at diagnosis; the lung is the most com-
mon metastatic site. This sarcoma is a relatively rare disease with limited therapeutic
options. The majority of patients are initially responsive to chemotherapy with vincristine,
doxorubicin (Adriamycin), and cyclophosphamide. However, relapsed disease is usually
extremely difficult to treat because of its resistance to chemotherapy (Zhou et al., 2001).
286 Chapter 12
Clinical studies indicate that overexpression of HER-2 correlates with poor prognosis
and shorter patient survival in osteosarcoma (Onda et al., 1995). There is a close associa-
tion between HER-2 overexpression and resistance to chemotherapeutic agents (Tsai et al.,
1995). Zhou et al. (2001) have investigated HER-2 expression in three different human
Ewing’s sarcoma cell lines (TC71, RD, and A4573). It was found that this protein is over-
expressed in the three cell lines. This study also indicates that E1A gene therapy may pro-
vide a new approach for treatment of this carcinoma. It has been demonstrated that
transduction of TC71 cells with the ElA gene using an adenoviral vector downregulates
HER-2 overexpression in these cell lines (Zhou et al., 2001). Downregulation of HER-2
increases apoptosis in tumor cells that overexpress the oncogene.
Ovarian Carcinoma
Prostate Carcinoma
Prostate carcinoma is the most common cancer in men in the United States.
Approximately 180,400 new cases were diagnosed in 2000, with 31,900 deaths as a result
of the disease. Prostate cancers typically begin with androgen-dependent lesions, but the
cancer usually becomes androgen-independent when the disease is advanced. It has been
shown, for example, that androgen-independent LAPC-4 prostate cancer sublines express
a higher level of HER-2 than that expressed by their androgen-dependent counterparts
(Carter et al., 2001).
Alterations in the signal transduction pathways mediated by HER-2 play an impor-
tant role in the progression of prostate cancer. This protein is normally expressed at a very
low level in a few human secretory epithelial cells but is overexpressed in a number of
human cancers, including prostate carcinoma. Overexpression of HER-2 is an indicator of
poor prognosis in prostate cancer patients.
Because of the deaths and considerable clinical complications that occur as a result
of prostate cancer, metastatic forms of the disease have been and remain an important tar-
get for novel therapeutic interventions. Although standard hormonal therapy for metastatic
prostate cancer has a high response rate (70–80%), hormone resistance ultimately devel-
ops, which necessitates additional therapy. Novel therapeutic HER-2-specific immuno-
toxins, such as scFv (FRPS)-ETA, have been developed and might be useful agents for the
treatment of prostate cancers with high levels of HER-2 protein. Another example is the
development of bispecific antibodies (e.g., MDXH210) designed to direct the cytotoxic
effects of monocytes and macrophages to destroy tumor cells expressing HER-2. These
constructs are discussed elsewhere in this chapter.
applying the ribonuclease protection assay, and at the DNA level using the Southern blot
and hybridization method. Activation of proto-oncogenes can be determined by assessing
the level of RNA expression or DNA amplification. A large number of patients with squa-
mous cell carcinoma of the cervix were recruited. EGFR showed a high percentage
(74.2%) of overexpression with immunohistochemical staining and 35.4% of DNA ampli-
fication in squamous cell carcinoma of the cervix. In contrast, HER-2 showed only 19 8%
of overexpression with staining and 17.2% of DNA amplification. It is concluded that the
abnormal expression of these two proteins has no prognostic significance on survival rates.
A number of methods are available for analyzing tumor HER-2 status. The selection
of the method depends on the target molecule to be detected. The target molecules are DNA
mRNA, and protein (Fig. 12.2). HER-2 gene amplification can be detected by Southern blot
(Press et al., 1994), slot blot (Naber et al., 1990), and dot blot assays (Descotes et al., 1993),
fluorescence in situ hybridization (FISH) (Persons et al., 1997), in situ hybridization (ISH)
on isolated nuclei or tissue sections (Smith et al., 1994), and polymerase chain reaction
(Gramlich et al., 1994). Assays to determine mRNA overexpression include Northern blot
(Slamon et al., 1989), Western blot (Press et al., 1994), slot blot (Naber et al., 1990) and
ISH (Naber et al., 1990). Methods to assess HER-2/neu protein product overexpression
290 Chapter 12
include Western blot analysis (Slamon et al., 1989), immunoassays (Dittadi et al., 1997),
FISH (Lebeau et al., 2001), and immunohistochemistry (Pauletti et al., 2000).
Some of the above-mentioned methods have limitations. Southern blot analysis
requires a large amount of high-quality DNA, and the results are unreliable if the sample
has a low percentage of tumor cells (Ross and Fletcher, 1998). For these and other draw-
backs, this method is precluded from becoming a routine clinical protocol to determine
HER-2 status.
Polymerase chain reaction (PCR), on the other hand, has several advantages: it can
be used to analyze small numbers of tumor cells, DNA from formalin-fixed, paraffin-
embedded tumor tissue can be used, and it can be automated and standardized. Quantitative
PCR techniques are currently being assessed for their clinical application to HER-2 DNA
testing (Vona et al., 1999). However, presently the PCR technology is not optimally suited
for routine, clinical application (see next section for details of quantitative analysis of
HER-2/neu expression).
It is apparent that many of the above-mentioned methods to analyze gene amplifica-
tion and mRNA overexpression are beyond the scope of most pathology laboratories for
technical reasons, and most of these assays require prospective collection of fresh tissue
and thus are not applicable to archival tissue specimens.
Fluorescence in situ hybridization is more widely used than Southern blot and PCR
techniques and allows visualization of HER-2 DNA in individual cells using a specific
fluorescence-labeled probe. Recently, Pauletti et al. (2000) have demonstrated HER-2/neu
gene amplification in infiltrating breast adenocarcinoma using FISH with a HER-2/neu-
specific probe. Another elegant study has demonstrated three-color FISH images of breast
carcinoma cell nuclei from two primary tumors with HER-2 oncogene amplification
(Järvinen and Liu, 2000). By performing dual-color FISH, the HER-2/neu gene status can
be quantified, and information about the ploidy status of chromosome 17 can also be
obtained (Walch et al., 2001). The detection of gene amplification in individual tumor cells
is one of its major advantages. However, in the absence of standardization, results will dif-
fer. For details about detecting HER-2/neu amplification with FISH, see Ross et al. (2001).
This detection method in breast cancer is based on the Oncor INFORM HER-2/neu Gene
System using a sequence biotinylated probe (Oncor, Gaithersburg, MD).
To cope with the above-mentioned problem, quantitative gene expression analysis can
be accomplished in combination with laser-assisted microdissection in formalin-fixed,
paraffin-embedded tissues. Using an optimized RNA microscale extraction procedure in
conjunction with real-time quantitative reverse transcriptase-polymerase chain reaction
(QRT-PCR) based on fluorogenic TaqMan methodology, Specht et al. (2001) have ana-
lyzed the expression of a panel of cancer-relevant genes, including HER-2/neu. They used
54 microdissected nonneoplastic and neoplastic archival samples from patients with
Barrett’s esophageal adenocarcinoma for analyzing HER-2/neu expression with the QRT-PCR
and compared it with that obtained in parallel with fluorescence in situ hybridization and
immunohistochemistry. The results of these three methods matched one another.
The QRT-PCR has been used for comparing quantitative mRNA expression of
HER-2/neu with DNA and oncoprotein levels in the metaplasia-dysplasia-adenomacarci-
noma sequence of Barrett’s adenocarcinoma (Walch et al., 2001). Tissue sections from the
same area can be used for QRT-PCR of laser-microdissected tumor cells and for immuno-
histochemistry. The method has been successful in quantifying HER-2/neu gene expres-
sion in small microdissected tissue samples from archival material as well as in premalignant
lesions (Specht et al., 2001; Walch et al., 2001). Only a locus-specific HER-2/neu gene
amplification is associated with strong mRNA overexpression and strong plasma mem-
branous immunostaining in Barrett’s adenocarcinoma. Quantitative PCR techniques are
currently being assessed for their clinical application to HER-2 DNA testing (Vona et al.,
1999). However, at present PCR technology is not optimally suited for routine, clinical
application.
The QRT-PCR is a sensitive, accurate, and highly reproducible method for studying
gene expression. The method is based on the 5' nuclease activity of Taq DNA polymerase
and involves cleavage of a specific fluorogenic hybridization probe that is flanked by PCR
primers spanning an amplicon range of 60–150bp (Gibson et al., 1996). It is capable of
detecting PCR products as they accumulate during amplification. The reactions are char-
acterized by the point during cycling when PCR amplification is still in the exponential
phase, allowing precise quantitation of RNA over a wide dynamic range. Because of the
small target size, this approach is suitable for quantitative determination of gene transcript
levels, even in tissue extracts containing partially fragmented RNA. Because only small
amounts of RNA are required, this technique is applicable to small clinical biopsies and
microdissected cell clusters from frozen or formalin-fixed, paraffin-embedded tissue sec-
tions (Specht et al., 2001). Laser-assisted microdissection is indispensable for the selective
analysis of stroma-free tumor cell populations, circumventing the problem of tissue het-
erogeneity as well as providing the possibility of assigning characteristic gene expression
patterns to particular histological phenotypes (Simone et al., 1998). This methodology
opens new avenues for the investigation and clinical validation of gene expression changes
in archival tissue specimens.
Three main techniques have been used for detecting HER-2 oncoprotein overexpression:
Western blot analysis, enzyme-linked immunosorption assay (ELISA), and immunohisto-
chemistry. Western blot analysis is limited to basic research rather than routine clinical
292 Chapter 12
analysis of HER-2 status because it requires fresh tumor homogenates. The use of tumor
homogenates results in large variations, depending on the amount of stromal and other
nontumor cells present in the homogenate, causing a potential dilution effect (Ross and
Fletcher, 1998).
Enzyme-linked immunosorption assay can be used to measure HER-2 protein in
tissue homogenates or in serum. It is a relatively simple technique and is well suited to
automation (van de Vijver, 2001). However, when tumor cytosolic fractions are used, his-
tological information is lost and invites the undesirable dilution effect. Therefore, the
ELISA assay is not used routinely to determine HER-2 status.
On the other hand, immunohistochemistry facilitates the identification of HER-2 protein
overexpression in individual tumor cells and has become the most common approach for
determining HER-2 status. Numerous reasons for its advantages are detailed throughout
this book. As is true in many other techniques, routine immunohistochemistry has the
disadvantage of being nonstandardized. One way to significantly minimize interlaboratory
variability and achieve a degree of standardization is to use a single commercial test kit
such as the HercepTest (Dako) discussed below. Another limitation of immunohistochem-
istry is that it relies on subjective interpretation of staining results. Many variables affect
staining results, which are discussed in Chapter 5 and elsewhere in this book.
Table 12.1 indicates one variable, i.e., antibodies, that affects HER-2 protein positiv-
ity. This table also indicates the role of different antibodies in determining HER-2 positiv-
ity in breast tumors, using immunohistochemistry.
In conclusion, immunohistochemistry and FISH are recommended for HER-2/neu
analysis in both routine clinical practice and in clinical research studies, using sections of
formalin-fixed, paraffin-embedded tissues. Although these two methods show a high level
of correlation with each other in the evaluation of HER-2/neu status of breast cancer,
immunohistochemistry is preferred for routine use. The FISH procedure requires more
time and expense than immunohistochemistry. In addition, the FISH slides must be stored
at –20°C or lower and are prone to quenching of the fluorescent signal with time. More
important, immunohistochemistry provides information on immunostaining as well as on
cell morphology of the same section. However, according to Pauletti et al. (2000), FISH
provides superior prognostic information in segregating high-risk from low-risk breast
cancers than that obtained with immunohistochemistry. Antibodies used in this procedure
Oncoprotein 293
are discussed later. Excellent studies comparing these two methods have been carried out
by Jacobs et al. (1999) and Lebeau et al. (2001).
Controversy continues over whether HER-2/neu gene amplification or gene product
p185 overexpression is the best predictor of therapy response and clinical outcome. Because
of this uncertainty, it is apparent that further clinicopathological studies are required to
demonstrate whether HER-2/neu amplification or protein overexpression, or a combination
of the two methods, has prognostic value in breast cancer. The most widely applied tech-
niques to detect the amplification of this gene and the overexpression of its protein product
are FISH and immunohistochemistry, respectively. The ideal approach is to correlate quan-
titative FISH with immunohistochemistry using antigen retrieval and computer-assisted
image analysis. The results are further improved when immunohistochemical studies are
assessed as the difference between tumor cells and nonneoplastic breast tissue. In other
words, immunostaining intensity of nonneoplastic breast tissue is subtracted from the neo-
plastic staining, correcting for cytoplasmic background staining. Only distinct membranous
immunostaining of HER-2 has prognostic value in breast cancer, while occasional cyto-
plasmic staining is without such relevance. Both techniques allow the study of small
amounts of formalin-fixed, paraffin-embedded tissue and the interpretation of the findings
on a cell-by-cell basis. Using this approach, Lehr et al. (2001) have convincingly demon-
strated a high degree of concordance between the two techniques in breast cancer.
BISPECIFIC ANTIBODIES
Bispecific antibodies are chemically or genetically linked antibodies with two het-
erologous antigenic specificities. Potentially antineoplastic bispecific antibodies can be
prepared by combining specificities for a tumor antigen and cytotoxic trigger molecules on
immunoeffector cells. Such studies were developed in order to target cytotoxic immuno-
effector cells to tumors to facilitate antibody-dependent cell toxicity (see also Chapter 2).
Essentially, bispecific antibodies (e.g., MDX-H210) are novel antibody constructs
designed to direct the cytotoxic effects of monocytes and macrophages to the destruction
of tumor cells expressing HER-2.
Recently, Sen et al. (2001) have described methods for generating highly effective
HER-2-specific cytotoxic T cells by arming activated T cells with anti-CD3 X anti-HER-2
bispecific antibody. In this method OKT3 and 9184 anti-HER-2 monoclonal antibodies
were conjugated and used to arm T cells that were subsequently tested for binding,
cytotoxicity, and cytokine secretion assays. Armed T cells aggregate and selectively kill
HER-2-positive breast cancer cells (MCF-7).
Such cytotoxic T cells can be produced by arming activated T cells with nanogram
quantities of OKT3/9184. Advantages of this technique are that arming activated T cells
with low doses of the bispecific antibody obviates the need for administering large amounts
of the antibody required for infusional therapy, and cytotoxicity is augmented by combin-
ing antibody targeting and T cell–mediated killing. Also, binding of effector cells at the
tumor site by armed activated T cells may augment tumoricidal activity, as well as increase
local cytokine secretion leading to recruitment of other immune effectors. This approach is
unique; in future clinical trials, billions of activated T cells could be armed with milligrams
of the bispecific antibody, thus becoming HER-2-specific cytotoxic T lymphocytes.
294 Chapter 12
VACCINES
Recently, there has been renewed interest in developing vaccines for use in cancer treat-
ment. The main factors giving impetus to this therapeutic approach include a better under-
standing of the immune system, the identification of several T cell–specific tumor
antigens, more effective adjuvants, and the ability to construct more immunogenic mole-
cules using recombinant DNA techniques (Murray et al., 2000). Current vaccine strategies
for the treatment of solid tumors tend to focus on the cellular arm of the immune response.
The overexpression of HER-2 protein in cancer cells makes it an ideal target for vac-
cines and other targeting strategies. Vaccines optimized to induce maximum T cell immu-
nity to HER-2 may lead to potent in vivo antitumor immunity. HER-2 protein has been
evaluated as a potential target for the development of cancer vaccines because preexistent
T cell and antibody responses to HER-2 have been described in breast cancer patients
(Disis and Cheever, 1996). In other words, breast cancer patients have preexisting immu-
nity to the HER-2 receptor in the form of elevated antibody titers and T cell immunity.
Elevated anti-HER-2 T cell responses have been demonstrated in breast and ovarian can-
cer patients following immunization with peptides derived from the HER-2 protein (Disis
et al., 1999). However, whether peptide-specific T cell responses can be translated to
antitumor immunity has yet to be established.
A recent study has utilized an in vivo murine tumor model expressing human HER-2
for evaluating potential HER-2 vaccines consisting of either full-length or variable subunits
of HER-2 delivered in either protein or plasmid DNA form (discussed later) (Foy et al.,
2001). The mechanism of protection elicited by plasmid DNA vaccination appears to be
exclusively CD4-dependent and not CD8- or antibody-dependent, whereas the protection
observed with intracellular domain protein vaccination requires both CD4 and CD8 T cells.
However, the exact mechanism(s) responsible for immunity to DNA has not been elucidated.
Another recent study supports the use of the dendritic cell-based vaccine as a thera-
peutic strategy to target both CD4 and CD8 T cells to HER-2 (Chen et al., 2001). To min-
imize the possibility of deleterious effects, the transforming activity of the HER-2
molecule can be inactivated by a single amino acid substitution (lysine to alanine), unlike
other studies in which the entire intracellular domain was removed.
Genetic Immunization
of MHC class I and/or class II molecules. Direct intramuscular injection with DNA plasmid
expressing HER-2/neu tends to induce antigen-specific cellular and humoral responses in
mice (Wei et al., 1999).
Chen et al. (1998) have shown that plasmid DNA encoding a truncated rat erbB-2/neu
that lacked the intracellular domain could induce protective immunity against erbB-2/
neu–expressing mammary tumors as effectively as plasmid encoding the full-length
erbB-2/neu oncogene. However, more recent clinical trials demonstrated that CD4 T cells
recognizing peptides from both extracellular and intracellular domains of human erbB-2/neu
protein can be induced by peptide vaccination. Vaccination with full-length DNA through
ex vivo targeting of the dendritic cells has the advantage of presenting the complete repertoire
of erbB-2/neu epitopes in association with MHC class I and class II molecules, maximizing
the number of peptide epitopes available for T cell recognition (Chen et al., 2001).
DNA vaccination requires host dendritic cells for priming the T cell response, either
through direct transfection or antigen transfer from transfected nonhematopoietic cells.
Because dendritic cells are functionally impaired in cancer patients, antigen processing
and presentation following genetic immunization may be inefficient. However, this prob-
lem can be overcome by differentiating ex vivo dendritic cells from precursors present in
the peripheral blood. The ex vivo cultured dendritic cells are fully functional and can be
used as cellular vectors for vaccines (Dhodapkar et al., 1999).
IMMUNOHISTOCHEMISTRY
extent) of the (HER-2/neu) gene product in breast cancer tissue (Kay et al., 1994). For
detecting this gene product in bladder, lung, and renal tumors, monoclonal antibody NCL-
CB11 has been used (Kay et al., 1994). This antibody recognizes the internal domain
(cytoplasmic staining) of the HER-2/neu gene product. The expression of this oncoprotein
has also been studied in mammary Paget’s disease using three different antibodies (Edorh
et al., 1995). Antibodies NCL-CB11 and NCL-CBE1 were used for recognizing internal
and external domains, respectively, of this protein.
As indicated above, a number of different monoclonal and polyclonal antibodies have
been used for determining the HER-2/neu status in breast cancer. A wide range of
HER-2/neu overexpression rates has been reported in different studies, varying from 9–60%.
This range resulted from using a wide variety of antibodies and/or variations in preparation
techniques, scoring criteria, and stage of cancer. The HercepTest containing rabbit-
polyclonal antibody A0485 (Dako) is superior to other antibodies. Recently, this antibody
was compared with five monclonal antibodies (9G6, 3B5, CB11, TAB 250, GSF-HER 2) and
one polyclonal antibody (A8010), using formalin-fixed, paraffin-embedded archival invasive
breast cancer tissues (Lebeau et al., 2001). This study has confirmed the superiority of A0485
over other antibodies. Another study also indicates the superiority of A0485 over polyclonal
antibody available from Oncor, Inc. (Gaithersburg, MD) (Maia, 1999). In conclusion, the
type of antibody used will affect the results of HER-2/neu protein immunohistochemistry.
Figure 12.3 shows the immunostaining of HER-2 oncoprotein in invasive breast carcinoma,
using monoclonal anti-c-erbB-2 antibody. Table 12.2 shows immunohistochemical localiza-
tion of this protein in different carcinomas in various tissue and organ types.
HERCEPTIN (TRASTUZUMAB)
A large body of evidence in the literature supports the idea that antibodies directed
against HER-2 can inhibit the growth of HER-2–expressing tumors through several mech-
anisms, including antibody-dependent, cell toxicity–mediated inhibition of HER-2 signal
transduction.
A number of murine monoclonal antibodies against the extracellular domain of the
HER-2 protein have been found to inhibit the proliferation of human cancer cells that over-
express HER-2 both in vitro and in vivo (Hudziak et al., 1989; Shepard et al., 1991). To
minimize immunogenicity, the antigen binding region of one of the more effective anti-
bodies (Herceptin, Ab4D5) was fused to the framework region of human IgG and tested
against breast cancer cells that overexpress HER-2 in vivo and in vitro (Carter et al., 1992;
Pietras et al., 1994). It is well established that Herceptin is superior to other antibodies
(e.g., polyconal antibody, Oncor, Inc.) and is called recombinant humanized anti-HER-2
antibody. Although Herceptin inhibits tumor growth when used alone, it has synergistic
effects when used in combination with chemotherapy.
Phase 1 clinical trials demonstrated that Herceptin is safe and confined to the tumor.
However, phase 2 trials indicated that the efficacy of the antibody is superior when given
with chemotherapy (Pegram et al., 1998). Phase 3 trials have indicated that Herceptin,
when added to conventional chemotherapy, can benefit patients with metastatic breast
cancer that overexpresses HER-2, prolonging relapse and overall survival (Slamon et al.,
2001). Similarly, extensive studies by Menden et al. (2001) support the weekly Docetaxel
298 Chapter 12
Oncoprotein 299
and Herceptin combination therapy for women with HER-2 overexpression metastatic
breast cancer. This combined treatment is thought to be well tolerated with significant anti-
tumor activity. However, a most troubling adverse effect of this antibody is cardiac dys-
function, especially when given concurrently with an anthracycline. The mechanism of the
cardiotoxicity of Herceptin is not known. Therefore, great caution is required in using this
antibody. Additional clinical trials are needed to further evaluate and confirm the usefulness
of Herceptin when used in combination with chemotherapy.
HERCEPTEST
consistent heating than do other heating methods. Any variation in the manufacturer’s
instructions can lead to variability in results.
Indeed, interobserver and interlaboratory variability of the interpretation of immunohis-
tochemical staining using the HercepTest is not uncommon for HER 1 + cases, because in
these cases it is not clinically relevant. In other words, scores of 0 and 1 + are considered neg-
ative and indicate probable nonresponsiveness to Herceptin. The variability is applicable for
HER 2+ and 3+ cases. The scoring systems and cutoffs used in different studies may vary.
Therefore, there is a need for optimizing the staining evaluation system. This variability can
be significantly minimized by quantifying HER-2 expression by image analysis (Press et al.,
1993). In a recent elegant study, Hatanaka et al. (2001) have quantified the levels of HER-2
protein expression in breast cancer using an image analyzing system and applied this system
for optimizing the interpretation of the HercepTest. By converting the quantitatively extracted
data into a scoring system based on the criteria, the outcome demonstrates a strong concor-
dance with the scoring data obtained from immunostaining. Figure 12.4 (Plate 6) shows the
image analysis of breast carcinoma immunostained with the HercepTest. In this system,
brown and blue signals are extracted and replaced with artificial green images.
A problem with the HercepTest arises when it is used at high altitudes. One of the
requirements of this test is that the slides must be heated in Epitope Retrieval Solution
(Dako) in a water bath at 95°C for 40 min. While this temperature can be easily obtained at
sea level, it is difficult to achieve at altitudes above 5,000 feet. At these altitudes, water boils
rapidly below 95°C as a result of air pressure on the boiling point. The use of boiling water
does not conform to the manufacturer’s directions. Various solutions to this problem include
extending the duration of heating to 50 min at 90°C or placing a metal lid over the water
bath. Any adjustment in the heating step should be assessed using the 1 + control cell line
included in the HercepTest kit. Ruegg and Lupfer (2001) have summarized various solutions
to this problem.
For the negative control, primary antibody is replaced with an irrelevant, isotype-
matched antibody. This step controls nonspecific binding of the secondary antibody. The
Oncoprotein 301
302 Chapter 12
positive control slides consist of sections of cell blocks of the three breast cancer cell lines
SKBR3, MDA-MB-175, and MDA-MB-231, which express 2.4 million, 92,000, and
22,000 HER-2 receptor molecules, respectively, by Scatchard analysis (Koeppen et al.,
2001). These receptor numbers correspond to immunohistochemical HER-2 scores of 3, 1,
and 0. These scores are defined by a lack of staining or membranous staining in less than
10% of the cells (0), incomplete membranous staining in more than 10% of the cells
(score 1), and complete membranous staining of strong intensity in more than 10% of the
cells (score 3). Scores of 2 or higher indicate HER-2 overexpression. A score of 2 or higher
is generally considered to be staining of 20–35% of the cells. Only membranous staining
should be considered as positive.
The incidence of HER-2 overexpression varies considerably in different tumors.
Table 12.4 shows an average score of HER-2 overexpression in representative human solid
tumors commonly evaluated in clinical practice. The specimens in this study consisted of
resections of primary tumors. Locally advanced and metastatic lesions of the same tumor
types may show significantly different incidences of HER-2 overexpression. Infiltrating
ductal carcinoma is one of the few tumor categories for which the incidence of HER-2
overexpression is consistent in most studies. These data were obtained using a single,
standardized immunohistochemical method (Herceptin).
Breast tumor tissues are fixed with formalin for 24 hr and embedded in paraffin, and
sections are deparaffinized according to standard procedures. They are heated in 0.1 mM
sodium citrate buffer (pH 6.0) in a water bath for 40 min at 95°C. After cooling for 20 min,
the sections are treated with 0.3% hydrogen peroxide containing 15 mM sodium azide for
Oncoprotein 303
10 min to block endogenous peroxidase activity. They are thoroughly rinsed in a washing
buffer (50 mM Tris-HCI buffered saline [pH 7.6] containing a detergent), incubated with
anti-HER-2/neu antibody for 30 min, and washed three times with the washing buffer.
The antibody, bound to HER-2 protein, is detected by incubation (for 30 min) with the
dextran polymer reagent conjugated with peroxidase and secondary antibody. Following
washing with the washing buffer, color development is achieved with DAB for 10 min in
an automatic stainer. As controls, slides containing three cell lines showing different HER-2
protein expression are included in the staining protocol. This step is used to confirm vali-
dation of the staining results.
Semiquantitative analysis is carried out based on a score of 0 (no staining or membrane
staining on less than 10% of tumor cells), 1+ (a faint perceptible staining), 2+ (a weak to
moderate staining of the entire membrane), and 3+ (a strong staining of the entire mem-
brane). Scores of 1+ to 3+ are also essential to be positive for more than 10% of tumor
cells. Scores of 0 or 1+ are negative for HER-2 protein overexpression, while scores of
2+ and 3+ are weak positive and strong positive, respectively. Only membrane staining is
evaluated.
Quantitative analysis of the HER-2/neu expression is calculated using a color video
camera with image processing software. The three images for analysis are picked up from
tumor lesions that exhibit predominant and typical features microscopically in each case
(Hatanaka et al., 2001). The quantitative labeling index for assessment is calculated by the
ratio of brown membranous staining area stained with DAB to round blue areas stained
with hematoxylin in consideration of tumor cell density in selected image. The extraction
of a brown or blue signal is carried out with specific protocols based on RGB color param-
eter, selected automatically in accordance with the protocols, and highlighted as artificial
green images (Fig. 12.4/Plate 6).
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Index
ABC method: see Avidin-biotin method Apoptotic cells, immunodetection of, 201, 202
AgNOR: see Nuclear organizer-associated Astrocytic tumor, HER-2
region location in, 285
Alkaline phosphatase anti-alkaline phosphatase, Autoclaving, 145, 146
89, 99 epitope retrieval with, 117
Aluminum chloride, role in antigen method of, 128, 129, 145, 148
retrieval, 76 Autostainers, 138, 153
Amyloid fibril protein, 123 Avidin-biotin method, 89, 90, 92, 98, 101
Androgen receptor, 83, 87, 107, 143, 144, 146
Angiogenesis, 20–25 Background staining, 133, 142, 146–148
in breast cancer, 22, 23 bcl-2, 136, 153, 154
in prostatic cancer, 22 Biotin, endogenous, 98–101
in vasculogenic mimicry, 21 role in background staining, 97
Antibody, dilution of, 80, 82 Biphasic mesothelioma, 25
penetration of, 79 Bispecific antibodies, 44–46, 293, 294
specificity of, 3 development of, 45, 46
Antibody-antigen interaction, mechanism of, Bladder tumor, 255
72, 73, 79, 82, 83 HER-2 location in, 285
Anticancer monoclonal antibodies, 47, 48 Blocking solution, 146
Anticancer vaccines: see Vaccines Boric acid, as antigen retrieval fluid, 76, 88, 135
Anti-cytokeratin 8 antibody, 118 Bouin’s fixative, 53, 59, 71, 96
Antigenicity, preservation of, 72 BrdU: see Bromodeoxyuridine index
Antigen retrieval, 1, 2 Breast carcinoma, 75, 107, 118, 123, 129, 135,
in archival tissues, 173, 174 138, 152, 210, 241, 263, 266, 269, 270,
in frozen brain tissue, 198, 199 282, 283, 301
general method of, 169–172 Bromodeoxyuridine index, 39, 40
with heat, 124
advantages of, 124 Calcineurin, 145, 153
mechanisms for, 117, 130, 131 Calcium ions, 117, 119, 147
Antigen retrieval fluids, 75–77, 143 modification of protein with, 120
molarity of, 74 role in antigen masking, 120–122
pH of, 74, 78 effect of pH, 122
Antigens, denaturation of, 72, 73 role of monoclonal antibodies, 122
Antivimentin antibody, 118 Carbohydrate antigens, 205–209
APAAP: see Alkaline phosphatase anti-alkaline retrieval with enzyme digestion, 208, 209
phosphatase retrieval with heat, 208
351
352 Index
CARD method: see Catalyzed reporter EnVision staining system, 99, 138–140
deposition procedure of, 138–140
Carnoy’s solution, 58, 59 Enzyme digestion for epitope retrieval, 117,
Catalyzed reporter deposition, 90, 92 118, 151–153
CD, definition, 44 method of, 152
Cell proliferation index: see Labeling index Enzyme-linked immunosorbent assay
Cell smears, immunostaining of, 182 (ELISA), microwave heat-assisted,
Chelating agents, epitope unmasking with, 120, 228–229
121 Epidermal growth factor, 22
Colon carcinoma, 85, 132 Epitope retrieval: see Antigen retrieval
Confocal scanning electron microscopy, Epitopes, 1, 2, 73
microwave heat-assisted, 230 EPOS staining system, 138, 139
Conventional oven, heating in, 175 Estrogen receptor alpha 265–267
Correlative microscopy, microwave Estrogen receptor beta 267–268
heat-assisted, 230, 231 Estrogen receptor gamma 268
Cross-reactivity, 144; see also Monoclonal Estrogen receptors, 74, 76–78, 85, 103, 106,
antibodies 130, 143, 153, 261–279
Cryopreservation, 65 antibodies for, 261–279
Cryosections, immunostaining of, 200, 201, distribution in carcinomas, 264
245 distribution in tissues, 268, 269
Cyclin D1 immunostaining, 184, 185 immunohistochemistry of, 273–275
Cytokeratin, 147, 154, 162, 176 immunostaining in prostate tissue,
277, 278
DAB as chromogen, 72, 105, 106 role in breast cancer, 269, 270
DAB-imidazol-copper sulfate, as chromogen, semiquantitative assessment of, 276, 277
98 Ewing’s sarcoma, HER-2 location in,
DCC: see Dextran-coated charcoal assay 285, 286
Detergents, 148–151
epitope retrieval with, 117, 118 False-negative staining, 60, 76, 80, 84, 102,
Dextran-coated charcoal assay, 5, 276 104, 105, 149
Diagnostic pathology, 1 False-positive staining, 10, 74, 78, 80, 97–99,
Digestive enzymes, 8 104, 105
DMP-30 accelerator, 160, 161 Fibronectin detection with monoclonal
Double immunofluorescence staining, 186, 187 antibodies, 114, 197
Double indirect immunofluorescence staining, Fine needle aspirates, 278
187–189 Fixation, advantages and limitations of, 53
Double immunostaining, 182–184 Flow cytometry, detection of antigens with,
Ductal carcinoma, 85, 184, 254, 271, 279 225–228
Fluorescence in situ hybridization, microwave
EDTA as antigen retrieval fluid, 76, 101, heat-assisted, 222
121–124, 153 gastrointestinal neoplasia, 222, 223
EDTA-NaOH as antigen retrieval fluid, 78 Formaldehyde, 53–60
Egg white, role in blocking endogenous biotin, effect of prolonged fixation with, 58, 59
100 fixation with, 57
Electron microscopy, 155–168 mechanism of fixation with, 54–56
ELISA: see Enzyme-linked immunosorbent nature of, 54
assay Formalin, 60
Endogenous calcium effect on antigen retrieval, overfixation with, 60
120–122 Formalin substitute fixatives, 59, 60
Endogenous peroxidase, 1, 146 Frozen sections, 136–139
Index 353