COLLEGE OF HEALTH SCIENCES

DIPLOMA IN MEDICAL LABORATORY TECHNOLOGY

YEAR 3 SEMESTER 1
SESSION 2021/2022

HISTOPATHOLOGY

PRACTICAL LABORATORY REPORT:

Enzyme Histochemistry and Immunohistochemistry

NAME : Thanashree Vignaeswaran

REGISTRATION NO. : MDC 190020
LECTURER : Encik Eidham bin Yusof
Objective
 Explain the procedure of enzyme histochemistry
immunohistochemistry staining.
 Perform the procedure of enzyme histochemistry and
Immunohistochemistry staining correctly and safely according to
standard operation procedure (SOP) provided
 Interpret and comment on the results of enzyme histochemistry
and immunohistochemistry staining.
Introduction
 Enzyme histochemistry is 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.
 Immunohistochemistry (IHC) is the most common application of
immunostaining. It involves the process of selectively identifying
antigens (proteins) in cells of a tissue section by exploiting the
principle of antibodies binding specifically to antigens in biological
tissues. Immunohistochemical technique is highly useful for
detection and quantification of target epitopes.
Immunohistochemistry (IHC) is a technique for identifying cellular
or tissue antigens by means of antigen-antibody interactions. In
more specifically, Immunohistochemistry staining, detect the
presence of specific protein markers that can assist with accurate
tumor classification and diagnosis. IHC has evolved to
complement the Hematoxylin & Eosin (H&E) and Special Stain
techniques that typically show tissue morphology (structure).
Materials and Apparatus
a. Enzyme histochemistry
 Fixed tissue section
 Tap water
 Incubation medium
 0.05% p-phenylene diamine dihydrochoride
 0.05M phosphate buffer
 1% osmium tetroxide
 Carrazzi hematoxylin
 Dehydrating agent
 DPX
b. Immunohistochemistry
 Tissue section
 TBS
 10% casein solution
 Diluted primary antibody
 Diluted biotinylated secondary antibody
 Labelled streptavidin
 DAB substrate solution
 Tap water
 Hematoxylin
 Dehydrating agent
 DPX

Procedure
a. Enzyme Histochemistry- Acetylcholinesterase
A) Preparation of tissue
- Cryostat sections of snap-frozen tissue cut at 10µm are air dried
and fixed for 30 seconds in 4% formaldehyde in 0.1M calcium
acetate.
- Frozen sections of formal calcium-gum sucrose treated blocks
of tissue.
B) Incubation medium
- Acetylthiocholine iodide 5mg
- 0.1M acetate buffer, pH6.0 (6.5ml)
- 0.1M sodium citrate 0.5ml
- 30mM copper sulfate 1ml
- Distilled water 1ml
- 4mM iso-octamethyl pyrophosphoramide
- Add 1.0ml 5mM potassium ferricyanide just before use.
C) Method
- Rinse the fixed sections for 10 seconds in tap water.
- Incubate at 37⁰C for 1 hour in the incubation medium.
- Wash briefly in tap water.
- Treat with 0.05% p-phenylene diamine dihydrochoride in 0.05M
phosphate buffer, pH6.8 for 45 minutes at room temperature.
- Wash in tap water.
- Treat with 1% osmium tetroxide for 10 minutes at room
temperature.
- Wash well in tap water, counterstain lightly (10 seconds) in
Carrazzi hematoxylin, wash, dehydrate, clear and mount in DPX
b. Immunohistochemistry
Labelled Streptavidin/streptavidin-biotin complex technique for
monoclonal antibodies
A) Monoclonal antibodies
- Rinse sections in TBS, then incubate in 10% casein solution for 10
minutes.
- Drain off excess casein.
- Incubate in optimally diluted primary antibody for 60 minutes.
- Wash slides in TBS.
- Incubate in optimally diluted biotinylated secondary antibody for 30
minutes.
- Wash slides in TBS.
- Incubate in optimally prepared labelled streptavidin or streptavidin-
biotin complex for 30 minutes. (When using streptavidin-biotin
complex, the reagents must be mixed 30 minutes before use in
order for complex to form).
- Wash slides in TBS.
- Incubate in DAB substrate solution.
- Wash in running water, counterstain in hematoxylin, dehydrate,
clear and mount.
Modification required for rabbit primary antibody
Step 5: Replace with biotinylated swine anti-rabbit secondary.
Results
A.Enzyme histochemistry- acetylcholinesterase

Increase in acetylcholinesterase
activity in parasympathetic nerve
fibers

Normal nerve plexus shows positive is

black staining in the cytoplasm with
negative staining of the nuclei of GC
which stand out as negative shadows

B) Immunohistochemistry- Streptavidin

Endogenous biotin in the mitochondria was labelled using streptavidin.

Discussion
 What is the principle of enzyme histochemistry? Enzyme
histochemistry is used to demonstrate the activity of enzymes
present on tissues. The visualization is based on the action of the
enzyme on a specific substrate. Following this reaction, an
insoluble product develops providing the location of enzyme.
 What are the importance of enzyme histochemistry?
Histochemistry is an important technique that is used for the
visualization of biological structures. As such, it is concerned with
the identification and distribution of various chemical components
of tissues through the use of stains, indicators as well as
microscopy.
 What are the DON’T’S in using Acetylcholinesterase? Fixation and
embedding of the tissue may affect the enzyme activity.
Embedding should be avoided because dehydration and high
temperature may damage the enzyme and its activity as well.

 What is the principle of immunohistochemistry?

Immunohistochemistry (IHC) is a method for detecting antigens or
haptens in cells of a tissue section by exploiting the principle of
antibodies binding specifically to antigens in biological tissues.
 How do you stain immunohistochemistry? IHC uses primary
antibodies to label a protein, then uses a secondary antibody
which is bound to the primary one.
 streptavidin is the better biotin-conjugate binder. Streptavidin lacks
any carbohydrate modification and has a near-neutral pH, it has
the advantage of much lower nonspecific binding than avidin.
Streptavidin will make sure the optimum fixation. To use
streptavidin properly for better results, choose antibody carefully.
Conclusion
 Acetylcholinesterase (AChE) histochemistry is a very useful
ancillary technique in the diagnosis and in aiding the operative
procedures of HD. It acts as a double check in the diagnosis of HD
(Hirschsprung’s disease).
 Streptavidin is a small bacterial protein that binds with high affinity
to the vitamin biotin. This streptavidin-biotin combination can be
used to link molecules such as radioisotopes and monoclonal
antibodies together. These bound products have the property of
being attracted to, and attaching to, cancer cells, rather than
normal cells. The radiolabeled products are more easily removed
from the body, thus decreasing their toxicity.