Name: Student ID:

Major: Paper: Immuno-Histochemistry

Grade: Teacher:

Q 1. Describe the PAS procedure to show glycogens in liver?

Periodic Acid Schiff (PAS) - is a staining method used to detect polysaccharides such as
glycogen, and muco substances such as glycoprotein, glycolipids and mucins in tissues.
PAS is combination of Periodic acid and Schiff Agent. PA is referred to Periodic Acid
which is made of Orthoperiodic acid & Meraperiodic acid

PAS - McMANNUS' PERIODIC ACID SCHIFF'S – GLYCOGEN

PURPOSE:
Glycogen is present in skin, liver, parathyroid glands and skeletal and cardiac muscle.
The PAS stain is used for demonstration of basement membranes, fungus secreting
adenocarcinoma from undifferentiated squamous cell carcinoma, and mucosubstances
secreted from the epithelia of various organs. A routine stain for liver and kidney
biopsies.
PRINCIPLE:
The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to
carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the
magenta color.
CONTROL:
For staining fungus; use a known positive such as those used for the GMS. Use skin,
aorta or normal liver for positive PAS staining.
FIXATIVE:
Any well fixed tissue.
TECHNIQUE:
Cut paraffin sections 4-5 m( 3m for kidney biopsies).
EQUIPMENT:
Rinse glassware in DI water. Coplin jar, microwave oven.
REAGENTS:
0.5% Periodic Acid:
Periodic acid 0.5 gm
Distilled water 100.0 ml
Mix well, label with date and
initial. Stable for 1 year.
Caution: Avoid contact and inhalation.
PAS STAIN
Lillie's Cold Schiff's Reagent:
Basic fuchsin 10.0 gm
Sodium metabisulfite 18.0 gm
Distilled water 1000 0 ml
Hydrochloric acid 10.0 ml

Stir solution for 2 hours, set in a dark cool place overnight. The solution is now a clear
light brown to yellow color. Add: Activated charcoal 500.0 gm (or two heaping spoons)
Stir. Filter through Whatman #2 filter paper into a 1000 ml graduated cylinder. Change
the filter paper often. Restore volume to 1000 ml with distilled water. Store in
Refrigerator, solution is stable for 6 months.

CAUTION: Carcinogen, corrosive.

SAFETY:
Wear gloves, goggles and lab coat. Avoid contact and inhalation. Hydrochloric acid:
Strong irritant to skin, eyes and respiratory system. Target organ effects via inhalation
on skin, respiratory, reproductive and fetal systems. Corrosive.
Schiff’s Reagent: Use extreme caution, Basic fuchsin (pararosaniline) is a known
carcinogen. Wear gloves, goggles, particle mask and lab coat, while preparing solution.
Work under the hood, keep hot, uncapped, solutions under the hood.
CARBOHYDRATES

PAS STAIN
PROCEDURE:
1. Deparaffinize and hydrate to distilled water.
2. Place slides into 0.5% Periodic acid for 5 minutes.
3. Rinse in distilled water.
4. *Schiff's Reagent, microwave HIGH power, for 45 - 60 seconds, until
deep magenta.
5. Wash in running tap water for 5 minutes.
6. Counterstain in hematoxylin for 3 minutes.
7. Wash in tap water, blue hematoxylin, rinse in distilled water.
8. Dehydrate in alcohol, clear, and coverslip.
* Conventional method: Schiff's Reagent, room temperature for 30
minutes.
RESULTS:
Glycogen, fungus: magenta
Nuclei blue
NOTES:
1. To check stain, pour 10 ml of formaldehyde in cylinder, add a few
drops of Schiff's, it should turn red-purple immediately.
2. Discard Schiff's if it turns pink while sitting in the refrigerator.
3. The white precipitate at the bottom of the Schiff's can be redissolved
if desired by gently warming and stirring the solution.
Rat liver. Stained with Periodic Acid-Schiff's reagent.
Q2. What are the characteristics of frequently used fixatives in Histochemistry?

Fixation:
This is the process by which the constitutes of cells and tissues are fixed in a physical
and chemical state so that they can withstand subsequent treatment with various
reagent with minimum loss of architecture this is done by exposing the tissue with
various chemicals named as fixatives.

Fixation is very critical steps in the fields of pathology, histology & cell biology by which
biological tissues are preserved from the decay. Fixation is essentially exists to prevent
the autolysis and degradation of the tissue and tissue components such that they can
be observed both anatomically and microscopically following sectioning.
Fixation is usually the first stage in a multistep process to prepare a sample of biological
material for microscopy or other analysis. A number of fixatives exist but formaldehyde
has been using over a century. Formaldehyde-mediated tissue fixation is thought to be
dependent on the formation of protein-protein and protein-nucleic acid cross-links.and
others has only been created in the last 10 years. Formaldehyde is not universal fixative
and others have only been created in the last 10 years.

Classification of Fixatives:
A) Crosslinking fixatives – aldehydes.
eg.formaldehyde,paraformaldehyde,gluteraldehyde,adipaldehyde,malonaldehyde
etc.
B) Precipitating/Denaturating fixatives.
eg.Ethanol,Methanol, acetone and Acetic acid etc.
C) Oxidising Agents.
eg.Osmium tetroxide, Potassium dichromate, andpotassium permanganateetc
D) Others.
a) Mercurials( Mercury perchloride, B-5 and Zenker's) etc.
b) Picrateseg. Picric Acid
Frequently Used Fixatives:
A. Single Fixative B. Mixed Fixatives.

A. Single Fixatives:
1) Formaldehyde
2) Glutaraldehyde :
3) Acetic acid :
4) Acetone
5) Methyl alcohol , ethanol
6) Picric acid :
7) Potassium dichromate :
8) Osmium tetroxide
1) Formaldehyde:
 Merits (Advantages):
i. It is cheap, readily available, easy to prepare and relatively stable.
ii. It penetrates tissue well.
iii. It does not over hardens tissues.
iv. It preserves fat and mucin.
v. It is the best fixative for the nervous tissue.
Shortcomings (Disadvantages):
i. The solution is irritating to the skin causing allergic dermatitis.
ii. Fumes are irritating to eyes and nostrils causing allergic rhinitis and
sinusitis.
iii. It may produce considerable shrinkage of tissues.
iv. Formalin reduces both the basophilic and eosinophilic staining of cells.
v. It forms abundant brown artifacts, pigments and granules.
2) Glutaraldehyde :
It is an aldehyde M.W. 100 or 2 formaldehyde residues linked by a straight 3
carbon chains. A 2.5% solution is best used for small fragments and small
needle biopsies, fixed adequately for 2-4hours in room temperature. A 4%
solution is recommended for large tissue not exceeding 4mm. in thickness
for 12-24 hours at room temperature.
Merits (Advantages):
i. It has a stable nature, stable effect on tissues, giving a firmer texture and
color.
ii. It preserves cellular and plasma protein better.
iii. Best used for EM where a high degree of cytological preservation is
paramount.
Shortcomings (Disadvantages):
i. It is more expensive.
ii. It is less stable.
iii. 5% and more gluteraldehyde causes tissue contraction.
iv. Not suitable for immunochemical staining because of its antigen
masking property.
3) Acetic acid :
Acetic Acid Fixative are used at ice-cold temperature (-40 degree Celsius)
Merits (Advantages):
i. It fixes and precipitates nucleoprotein.
ii. It precipitates chromosomes and chromatin materials, which makes
it useful in nuclear studies.
Shortcomings (Disadvantages):
i. It causes considerable swelling of tissues.
ii. It destroys mitochondria and golgi elements.

4) Acetone:
Acetone used at ice-cold temperature (-40 degree Celsius) used only in enzyme
studies.
Merits (Advantages):
i. It is recommended for phosphatases and lipases studies.
 ii. It is used for fixing brain tissues for rabies.
Shortcomings (Disadvantages):
I. It evaporates rapidly.
II. It dissolves fats.
III. It causes tissue contraction.
5) Alcohol Fixatives:
Are used for rapid denaturing and precipitation of proteins by destroying
hydrogen and other bonds. Itmust be used in concentration from 20-100%
because lesser concentrations lyse the cells.
Merits (Advantages):
i. Ideal for smaller tissues fragments.
ii. Excellent for glycogen preservation.
Shortcomings (Disadvantages):
I. It causes lysis of red blood cells.
II. It dissolves lipids and fats.
III. It causes polarization of glycogen.
IV. It causes tissue contraction
6) Picric Acid Fixatives:
It is usually used in strong or saturated solution. The solubility of picric acid is that
1.7 grams will justdissolve in 100mL of distilled water at 15 degree
Celsius. Chemically, picric acid is 2.4,6-trinitrophenol.
Merits (Advantages):
i. It is the best fixative for glycogen.
ii. It penetrates tissues rapidly.
Shortcomings (Disadvantages):
i. It lyses red blood cells.
ii. It causes considerate shrinkage of tissues.
iii. Picric acid is potentially explosive.
7) Potassium Dichromate:
Used in 3% aqueous solution. It is a strong fixative for certain lipids.
Merits (Advantages):
i. It fixes but does not precipitate cytoplasmic contents.
ii. It preserves mitochondria.
Shortcomings (Disadvantages):
i. It penetrates tissues slowly.
8) Osmium Tetroxide Fixatives:
Commonly known as osmic acid. It is pale yellow. It dissolves in water (up to
about 6% at 20 degreeCelsius) forming a solution which is a strong
oxidizing agent.
Merits (Advantages):
i. It fixes conjugate fats and lipids permanently by making them
insoluble during subsequenttreatment with alcohol and xylem.
ii. It produces excellent nuclear staining.
Shortcomings (Disadvantages):
I. It is very expensive.
II. It is a slow fixing agent suitable only for small tissues.
 III. It causes tissue expansion

B. Mixed Fixatives:
Because no single fixative is ideal for the preservation of all tissue
components, mixtures of fixatives have been tried in an attempt to
compensate the shortcomings of one by another.

a. Carnoy:
Ethanol: Chloroform : ethylic acid =6:3:1, use when fresh. Suitable for nucleus
acid, glycogen, immune organs and glands.
b. Paraformaldehyde -glutaraldehyde :10% paraformaldehyde 10ml,
25% glutaraldehyde 5ml, pH7.4 0. 2M phosphate buffer 25ml,
DDH2O10ml. Suitable for microstructures, enzyme histochemistry and
immunohistochemistry.
c. Bouin:
Picric acid saturated water solution: formaldehyde : glacial acetic acid =
15:5:1, use when fresh. Mostly used.

The way to Select a Fixative:

In most established laboratories a routine fixative or fixatives have already been chosen
and used for a considerable time on a range of specimen types. Most frequently the
routine fixative will be neutral buffered formalin with other agents used for bone marrow
trephines (perhaps a zinc formalin), renal biopsies, frozen sections etc. Buffered
formalin is widely used because it is probably the most flexible of agents. It can be
incorporated into the processing schedule on enclosed tissue processors. It permits the
successful application of a wide range of special stains. Immunohistochemistry
methods, that generally include an antigen retrieval step, have been optimized for
formalin-fixed tissues, and tissue specimens can be stored in formalin for extended
periods without major deleterious effects.
Some laboratories are currently looking to replace formalin with a less toxic reagent. In
a research environment, where a specific tissue element is being studied, there may be
more control over the fixation step, and it is worthwhile testing several reagents before
making a final decision under the microscope:

• Toxicity of the fixative (both short-term and cumulative)

• Volatility of its components and the equipment available to prevent
staff exposure to the agent
• Flammability
• The effect of over-fixation on tissues (does prolonged fixation
damage tissues?)
 • Storage requirements if specimens cannot be left in fixative
• The compatibility of the fixative with your tissue processor (might it
damage components?)
• The practical and legal requirements of disposal after use

Changing to a different fixative requires very careful consideration and thorough

evaluation.

Q-3:Describe the procedures to show alkaline phosphatases (AKP) in kidney from

the very beginning.
Alkaline phosphatase is an enzyme found in your bloodstream. ALP helps break down
proteins in the body and exists in different forms, depending on where it originates. It is
mostly produced in your liver, but some is also made in your bones, intestines, and
kidneys. In pregnant women, ALP is made in the placenta

Humans and most other mammals contain the following alkaline phosphatase
isozymes:
ALPI – intestinal (molecular weight of 150 kDa)
ALPL – tissue-nonspecific (liver/bone/kidney)
ALPP – placental (Regan isozyme)

Elevated levels of Alkaline Phosphatase

If it is unclear why alkaline phosphatase is elevated, isoenzyme studies using
electrophoresis can confirm the source of the ALP. Heat stability also distinguishes bone
and liver isoenzymes[citation needed] ("bone burns, liver lasts"). Placental alkaline
phosphatase is elevated in seminomas and active forms of rickets, as well as in the
following diseases and conditions:
• Biliary obstruction
• Bone conditions
• Osteoblastic bone tumors
• Osteomalacia
• Liver disease or hepatitis
• Leukemia
• Lymphoma
• Paget's disease
• Sarcoidosis
• Hyperthyroidism
• Hyperparathyroidism
• Pregnancy

Lowered levels-
The following conditions or diseases may lead to reduced levels of alkaline
phosphatase:
• Hypophosphatasia, an autosomal recessive disease
• Postmenopausal women receiving estrogen therapy because of
osteoporosis
• Men with recent heart surgery, malnutrition, magnesium deficiency,
hypothyroidism, or severe anemia
• Children with achondroplasia and cretinism
• Children after a severe episode of enteritis
 • Pernicious anemia
• Aplastic anemia
• Chronic myelogenous leukemia
• Wilson's disease
• Oral contraceptives

Process to find AKP in the kidney:

There are 2 process to find AKP

1. Alkaline phosphatase (AKP) metal deposition method

2. Lead Phosphate { Pb3 (PO4)2 } method.

Here we are going to discuss the Num 1 method:

Process:

1. Theory:
AKP is One kind of hydrolase . Activated by manganese ion(Mn 2+), magnesium
ion(Mg2+), zinc ion (Zn2+)and cobalt ion (Co2+), and inhibited by cysteine , cyanide (CN-),
arsenate and tetramisole. At pH 9.2, AKP hydrolyze sodium glycerophosphate to
produce phosphate radical (PO43-), which will react with Ca2+, then replaced by Co2+.
When encountered with sulfur ion(S2-), the black deposit cobalt sulfide (CoS) is formed.

2. Staining:
a. Incubation media : pH9.2.
0. 2% Barbital sodium 6ml.
0. 2% Sodium glycerophosphate 6ml.
0. 2% Calcium nitrate 3ml.
DDH2O 15ml

Procedure :
Paraffin-embedded Wash in water. 2%
Incubation
tissue is de-waxed cobalt nitrate
media,37℃, 30min
to water. ( Co(NO3)2),3min.

Water wash,
Water wash. 1%
dehydration,
ammonium sulfide (
clearance and
(NH4)2S),2min.
reservation

Result: AKP positive position shows brown-black color

AKP metal deposition method, rat small intestine
AKP staining in kidney
Q.4 In the double staining technology of Immunohistochemistry, two kinds of
primary antibodies come from animals of same species, and the same enzyme is
used to show the two staining results. Do we need to wash up before the second
staining? Explain why and how to proceed the wash up?

In the double staining technology of Immunohistochemistry, two kinds of primary

antibodies come from animals of same species, and the same enzyme is used to shown
the two staining results, yes we need to wash up before the second staining.
HRP is shown by CN and DAB, respectively. The CN-staining result and/or antibodies
need to be washed up, and then the second antigen is detected.

Wash up methods:
a. Oxidation method: 2.5% KMnO4: 5% H2SO4: DDH2O=1:4:10~260, 1min, to remove
antibodies. 0.5% sodium metabisulfite (Na2S2O5) is used to bleach slide, and CN is
used for first demonstration.
b. Acid solution method: After CN demonstration and pictures are taken, the ethanol
is used to dissolve its blue color. The treatment with pH2.2, glycine - HCl buffer
containing dimethylfomamide (DMF) for 1~4 hours will destroy antigen-antibody
complex.
c. Formaldehyde vapor method: 1 L hermetic container + 3g paraformaldehyde, 80℃,
1~4 hours, to destroy the antibody binding sites.

Wash up procedures A:
After first staining, the antibody and
enzymes are removed, but the final
productions are preserved. PAP staining
and CN-H2O2 visualization methods are
used. After TBS wash, Oxidation method
is used to wash up. Then a TBS wash
again and then PAP method staining.0.05%DAB-0.01%H2O2 is used for visualization.
Again a TBS wash and then Glycerol reservation.

Wash up procedures B:
After first staining, the observation fields
and results are recorded by camera. The
organic solvents and oxidation method
are used to remove the final colorful
production, antibodies and enzymes.
Then the second staining is carried on.
The same observation fields and structures are recorded by camera again. Then PAP
method staining and CN-H2O2 visualization. After TBS wash, TBS reservation and take
pictures. Again a TBS wash. Then Alcohol-xylene-alcohol washes to remove blue,
DDH2O wash. Oxidation method to wash up. Again a TBS wash. Then PAP method
staining, 0.05%DAB-0.01%H2O2 visualization, a TBS wash and finally Glycerol
reservation - Find the same structure and take pictures.

Double Immuno-histochemistry Technique For Histological Specimen

● Four micro meter thick tissue sections with microtome are taken and

mounted on silane-coated slides from the selected blocks that were fixed

in 10% formalin.

● Sections are deparaffinized and rehydrated by alcohol before staining.

● Tissue specimens is treated with 3% hydrogen peroxide for 10 min to

quench endogenous peroxidase activity.

● Epitope retrieval is performed for both desired antibody as recommended

in 10mM citrate buffer.

● Protein blocking agent ready to use serum is applied for 10 minutes.

● Primary antibody at 25º C for 1 hour. Desired immunomarkers are used

● Wash in PBS buffer.

● Secondary biotinylated antibody ready to use for 10 minutes

● Wash in PBS buffer

● Streptavidin- peroxidase ready to use for 10 minutes

● Wash in PBS buffer

● DAB chromogen Invitrogen for 10 minutes

 ● Wash in running water

● Counter stain with haematoxylin and eosin

Q.5 beside what you studied in class, what are the new developments of
Histochemistry?

Over the past decade, considerable efforts to understand the states of specific gene
expression at cellular and/or subcellular levels have been made. For this particular
purpose, nonradioactive in situ hybridization to localize mRNAs has been developed
and improved substantially, and it is now recognized as a powerful, established light-
microscopical technique. In this review, we focus on the recent advances in the
technological aspects of nonradioactive in situ hybridization including the use of
synthetic oligodeoxynucleotide probes, the progress in analysis of signals, and the
application to electron microscopy. Also, southwestern histochemistry, a relatively new
method of localizing transcription regulatory proteins by utilizing haptenized DNA with
responsive element sequences is described. Then we discuss what we can see by
combining these molecular histochemical methods which were brought about by the
merger of molecular biology and structural biology.
Enzyme histochemistry serves as a link between biochemistry and morphology. It is
based on metabolization of a substrate provided to a tissue enzyme in its orthotopic
localization. Visualization is accomplished with an insoluble dye product. It is a sensitive
dynamic technique that mirrors even early metabolic imbalance of a pathological tissue
lesion, combined with the advantage of histotopographic enzyme localization. With the
advent of immunohistochemistry and DNA-oriented molecular pathology techniques, the
potential of enzyme histochemistry currently tends to be underrecognized. This review
aims to draw attention to the broad range of applications of this simple, rapid and
inexpensive method. Alkaline phosphatase represents tissue barrier functions in brain
capillaries, duodenal enterocyte and proximal kidney tubule brush borders. Decrease in
enzyme histochemical alkaline phosphatase activity indicates serious functional
impairment. Enzyme histochemical increase in lysosomal acid phosphatase activity is
an early marker of ischemic tissue lesions. Over the last four decades,
acetylcholinesterase enzyme histochemistry has proven to be the gold standard for the
diagnosis of Hirschsprung disease and is one of the most commonly applied enzyme
histochemical methods today. Chloroacetate esterase and tartrate-resistant
phosphatase are both resistant to formalin fixation, EDTA decalcification and paraffin
embedding. Early enzyme histochemical insight into development of a pathologic tissue
lesion and evaluation of function and vitality of tissue enhance our understanding of the
pathophysiology of diseases. In this process, enzyme histochemistry constitutes a
valuable complement to conventional histology, immunohistochemistry and molecular
pathology for both diagnostic and experimental pathology.
The advanced techniques of histochemistry being used in surgical histopathology
laboratories include:
● Laser microsection (LMD)techniques have become routine toll for the procurement
of a purified population of cells obtained from a complex tissue section for
subsequent molecular analysis.
● Hybrid model integrating immunohistochemistry
● Fluorescence in situ hybridization scanning system and ultrasensitive
immunohistochemistry

● Skeletal muscle biopsy.

● Rapid and easy detection of ganglia and nerves in cases of suspected
Hirschsprung’s disease.
● Demonstration of specific lactase or sucrose deficiency in jejunal biopsies.
● Demonstration of mast cells & white cells of the myeloid series.
● Miscellaneous:

Skeletal muscle biopsy:

Application of enzyme histochemical methods to cryostat sections of unfixed skeletal
muscle shows the presence of different fiber types, and changes in the number, size
and relative proportions of different fibers which are valuable in establishing the
diagnosis. Of these are enzyme histochemical techniques. Following methods are used
routinely:
a) Adenosine triphosphatase: ATPase methods are used in combination to distinguish
between type1 and type 2 fibers, and to further subdivide the type 2 fibers into 2A, 2B
and 2C subtypes. This distinction is diagnostically important since some muscle
diseases have characteristic patterns of loss, atrophy or grouping of specific fiber types
or subtypes. Some types of structural fiber abnormality (e.g. periodic paralysis) are also
demonstrated by the ATP-ase methods.
b) NADH diaphorase: Demonstrates mitochondria and the fine detail of the
sarcoplasmic reticulum of the fiber. It is used to detect very minor or early structural
abnormality in the sarcoplasmic reticulum network of the fiber, as well as mitochondrial
abnormalities. E.g. Mitochondrial myopathies.
c) Phosphorylase: Also distinguish between type 1 and 2 fibers but fades very quickly.
It is used to exclude McArdle’s disease, a primary phosphorylase deficiency.
d) Acid phosphates or non-specific esterase: To identify macrophages in necrotic
fibers and abnormal lysosomal activity in muscle fibers.
e) Cholinesterase: To highlight atrophic fibers and to demonstrate intramuscular nerve
twigs.

Detection of nerves & ganglia in suspected Hirschsprung’s disease: In

Hirschsprung’s disease in children, a variable segment of the rectum and colon is
devoid of ganglionic cells. In the effected segment peristalsis is impossible and the large
bowel becomes obstructed. The diagnosis may be suspected clinically and
radiologically but requires histological confirmation, usually by the examination of one or
more suction biopsy specimens of rectal mucosa and submucosa.The biopsy sample is
orientated under dissecting microscope control so that sectioning will include mucosa
and submucosa,then snapfrozen at -170 C in isopentane cooled in liquid N2 and
sectioned in a cryostat. Preliminary sections are stained with H & E and the sub mucosa
examined for the presence of ganglia. If sufficient sub mucosa is present or if no ganglia
are seen after examination of a no. of H & E stained levels, then 2 or 3 sections are
stained by cholinesterase method to demonstrate the fine nerve twings in the mucosal
lamina propria.

Demonstration of specific lactase or sucrase deficiency in jejunal biopsies:

For the assessment of jejunal mucosal biopsies in suspected celiac disease, the
specimen can be examined under the dissecting microscope and the presence or
absence of villi noted. Paraffin sections stains with H & E are used to assess villous
height, gland hyperplasia and intensity of inflammatory cell infiltrate in the lamina
propria. Alternatively, the biopsy can be snapping frozen, sectioned in a cryostat, and H
& E stained for rapid diagnosis.
Advantages: An alkaline phosphates method can be applied; alkaline phosphates
activity resides on the enterocyte surface, is a sensitive marker of structural and
functional integrity of the mucosal absorptive cells. This is particularly useful in
assessing histological recovery. Acid phosphates demonstrate some of the inflammatory
cells in lamina propria, and also identify lysosomal activity in villous enterocytes and
glandular crypt epithelial cells.

Demonstration of mast cells & white cells of myeloid series: Chloroacetate

esterase techniques have recently been applied to formalin-fixed paraffin sections to
assist in the identification of tissue mast cells and myeloid white cells. Two methods are
suitable: 1. Fast blue RR method: which gives a vivid blue reaction product (particularly
intense in mast cell cytoplasm). 2. Pararosanilin method: which gives a pinkish-red
reaction product?

Miscellaneous:
Use of acid phosphatase in the identification of prostate carcinoma.eg.when the tumor
is infiltrating the colon or bladder wall, or in bone metastases.
Application of acid and alkaline phosphatase methods to cryostat sections of jejunal
mucosal biopsy specimens.
Use of alkaline phosphatase methods in vascular endothelial tumors.
Q.6 List some merits and shortcomings of any aspects of this course?

Merits
Keeping in mind the Phd programs we are enrolled in this has been a very interesting
and useful subject.
The content of the course is very informative as well as beneficial from a professional
point of view. It covers the purpose methodically and in an efficient manner.
The lecture slides are arranged in a good way, beginning from the very basic to highly
advance techniques as we move on towards the end of the syllabus.
Learning environment is congenial and teacher taught relationship is kept friendly which
in itself is advantageous to the students.

DeMerits/Suggestions to Improve
The students would have been able to extract more out of the lectures if the lectures
had been delivered in a proper Lecture room with multimedia on.
It would be more beneficial if arrangements were made to learn common histochemistry
techniques practically within the laboratory, along with the theoretical lectures.