IMMUNOSTAIN AND HISTOCHEMISTRY

DEFINITIONS
Immunostaining used to localize
specific molecules in tissues. It is based
on antibody-antigen recognition.
Antibody (Ab) also known as
immunoglobulin. IgG is the most
commonly used antibody in
immunostaining.
Antigen (Ag) a substance which
evokes the production of one or more
antibody.
Epitope structural part of the antigen
that reacts with the antibody

HISTORY
1941 Albert Coons first introduced the
use of immunofluorescence as initial
attempts to label antibodies but wasnt
successful because the labels werent
visible under the microscope.
1967 Nakane and Pierce developed
the Enzyme-labelled antibody
technique by labelling an antibody with
an enzyme.
1970 Sternberger discovered the
peroxidase-antiperoxidase (PAP)
method.
1990s Antigen retrieval; it increased
the detection of antigens and sensitivity
of methods.
It was known that the retrieval of
nonreactive antigens in formalin-fixed,
paraffin embedded tissues was possible
through heating sections in buffer
solutions.


IMMUNOHISTOCHEMISTRY (IHC)
combination of immunologic and
histochemical techniques which allow
phenotypic markers to be detected and
demonstrated under the microscope
antibodies are cross-linked to enzymes
which can produce a colored end
product
uses polyclonal, monoclonal,
fluorescent labeled or enzyme-labeled
antibodies
immuno = antibodies ; histo = tissue

Antibody Characteristics
1. Monoclonal - identical copies of the same
unique antibodies, recognizes only a single
epitope.
- highly specific
- intrinsic cross-reactivity to
non-target can be problematic.
- potential for epitope loss
resulting in loss of staining.
- unlimited supply.
2. Polyclonal - a mixture of multiple antibodies
recognizing different epitopes of the antigen.
- in general, does produce stronger
signal greater potential for false positive
staining due to Ab cross reacting to others.
- limited supply.

Tissue Preparation for IHC
tissue must be:
a. prepared in a cryostat section
b. fixed in absolute methanol or acetone
for a few second

purpose: prevent destruction of some
of the labile antigenic sites

Sample Preparation:
1. Fixation
To prevent bacterial decomposition
To prevent autolysis by rapidly
terminating the enzymatic/metabolic
activities
Used to immobilize antigens while
retaining cellular and subcellular
structures through cross-linking
methylene bridges and Schiff bases
between basic amino acid residues of
proteins.
Tissue fixation is optimized for
maintaining morphology/ histochemical
staining

Characteristics of Fixatives:
- Acts Rapidly
- Safe/easy to use
- Inexpensive
- Preserves cellular and tissue structures
without introducing artifacts
- Stabilizes tissue, allows for handling and
processing

*The ideal fixative does not exist.

Fixatives:
- Aldehydes (formaldehyde,
glutaraldeyde)
- Oxidizing agents, metallic ions and
complexes (osmium tetroxide, Chromic
acid, Zn)
- Protein denaturing agents (acetic acid,
methanol, ethanol)
- Poorly-Understood mechanisms
(mercuric chloride, picric acid)
- Other: microwave,vapor fixation
- Combinations of above

2. Embedding
(Frozen Section)
for quick processing and situations
where the antibody may not be
compatible with paraffin compounds
are typically used for direct or indirect
immunofluorescence detection
methods
thickness: 10 microns

(Paraffin)
is used for maximum tissue
preservation and makes a stronger
block for sectioning tissue
thickness: 3-5 microns
3. Retrieval of Masked Antigens
Unmasking the antigen is sometimes
necessary because paraffinization and strong
fixations may inhibit the primary antibodies
attachment to the antigen.

Proteolytic Enzyme Digestion
used in formalin fixed paraffin
sections
trypsin and protease are the
enzymes commonly used
useful for demonstrating heavy
chain immunoglobulins,
complement and specific
antigens (i.e. cytokeratin)

Microwave Antigen Retrieval
involves boiling of formalin-
fixed deparaffinized sections in
solutions such as:
a. 0.01 M-citrate buffer (pH 6.0)
b. EDTA (pH 8.0)
c. Tris EDTA (pH 9.9 or 10.0)

most satisfactory exposure time
on heat: 20 minutes


Pressure Cooking Retrieval Antigen
less time consuming
allows more consistent
recovery of many antigens

4. Immunostaining
used to localize specific molecules in
tissues. It is based on antibody-antigen
recognition.
Detection is commonly achieved using a
colorimetric (enzyme) reaction or a
fluorescent signal generated from an
ultraviolet source.

Purposes:
It determines when and where the
biomarker is expressed.
It helps confirm the genotype and
phenotype.
For co-expression or co-localization.
Cell type-specific biomarkers.

METHODS IN IHC
1. IHC Controls
used to test the specificity of the
antibodies involved and to avoid
misinterpretations due to false (+) or
false (-)

Positive Control
- a section that is known and
proven to contain the antigen in
question

Negative Control
-omits the primary antibody
from the staining schedule or
replacing the specific primary
antibody by an immunoglobulin
that is directed against an
unrelated antigen

Internal Tissue Control
- also called as "built-in control"
-eliminate the variable of tissue
fixation between specimens
and controls
-contains the target antigen

2, Enzyme Labeling
incubated with chromogen to produce a
stable colored reaction
optimal incubation time: 30-60 minutes
at room temperature

enzyme labeling of Ag with
horseradish peroxidase
staining with chromogen
mixture (i.e. diaminobenzidine
DAB)
insoluble dark brown end
product
3, Direct Technique of IHC
conjugates the primary antibody
directly to the label
advantage:
simple and quick
requires only one application of
the reagent

disadvantage:
less sensitive
has risk of not detecting small
amounts of Ag
4. Indirect Technique of IHC
two or three step procedure that
involves application of the
unconjugated primary antibody and a
labeled antibody directed against the
first antibody
advantage:
inexpensive
more sensitive
Horseradish peroxidase
-most commonly used enzyme for
indirect antibody complex techniques

5. Peroxidase-Antiperoxidase (PAP) Technique

exposes an unlabelled primary Ab to
the Ag in the sample and is followed by
removal of any excess primary
antibodies thus allowing secondary
antibody to be introduced in the sample
6. Avidin-Biotin Complex (ABC) Techniques

uses avidin
- derived from egg white
- high affinity for biotin
-low molecular
weight vitamin
that can be easily
conjugated to Ab
and enzyme markers
staining sequence:
primary antibody
biotylynated secondary
antibody
(strept) avidin-biotin-enzyme
complex or enzyme labeled
streptavidin
7. Labeled Streptavidin Avidin Biotin Technique
(LSAB) Procedure)

4-8 times more sensitive than the old
ABC method
replaced avidin to streptavidin
Staining sequence:
rabbit or mouse antibody
biotinylated anti-rabbit or anti-
mouse immunoglobulin
strepatvidin enzyme conjugate

References:
Gregorios, J. (2006) Histopathologic
Techniques 2nd ed.
Meeker, K. (2012) Immunostaining 101,
http://www.hopkinsmedicine.org/mcp/
PHENOCORE/CoursePDFs/2012/18_Me
eker_IHC_101_9pH.pdf
Boenisch T, et. al,(2009)
Immunohistochemical Staining
methods, 5th ed