Immunohistochemistry is one of the most advance techniques in immuno-staining. In this presentation is a brief introduction to what is IHC, the principle, procedure and applications.

1. Immunohistochemistry

2. Immunohistochemistry
Chemical based
reaction
The study of
human tissue
The sciences that
deals with
immune system
Immunohistochemistry is the science that combine the techniques of
chemistry and histology in the study of the chemical constitution of
tissue and cells.
It’s a mixture of Sciences!

3. Keywords
Antigen: is a foreign substance that stimulates antibody formation and has the ability to
bind to an antibody.
● They are commonly proteins or glycoproteins of high molecular weight.
● An antigen that elicits a strong immune response known as “Immunogenic”
● The site on an antigen to which a complementary antibody may react with is called
“epitope” or “antigenic determinant”

4. Keywords
Antibody: is an immunoglobulin capable of specific combination with the antigen that caused
its production.
● They are produced by B-Lymphocytes as a protection from invasion of foreign
substances such as bacteria, viruses … etc.
● Each B-Cell produces antibodies with a single specificity.
● This mature B-cell is then called a Plasma Cell.

5. Keywords
Antibody-antigen reaction : is an antibody bind to antigen through the variable regions
of the antibody.
● This bonding may be hydrophobic, ionic, van der waals or hydrogen bonding.
● The strength of the binding of an antibody to a specific antigen is called “affinity”.
● The rate of the reaction is affected by temperature and pH of buffers and
diluents used in IHC procedures.

6. Principle of IHC
Cellular antigen Added antibody
Immune complex
Immune complex
Fluorescent dye
/enzyme
Soluble coloured substance
Soluble coloured substance
Chromogen in
presence of
enzyme
Insoluble coloured substance

7. Procedure of IHC
Sample collection Tissue Fixation,
dewaxing and
rehydration.
Antigen Retrieval Background Blocking
Application of
primary antibody
Application of
secondary antibody
Application of
substrate
Application of
chromogen

8. Cont.
Counterstain
Dehydration,
clearing and
mounting
Visualize the
sample
under the
microscope

9. Visualization Techniques
Immuno
Enzyme
Histochemistry
Immuno-
fluorescence
Histochemistry
➔ Fluorescence attached to antibody.
➔ Fluorescent microscope.
➔ Less frequently used.
➔ Enzyme attached to antibody.
➔ Light microscope.
➔ Most commonly used.

10. Tissue Fixation
4% paraformaldehyde in 0.1M
phosphate buffer.
2% paraformaldehyde with 0.2%
picric acid in 0.1M phosphate
buffer.
4% paraformaldehyde with 0.2%
periodate and 1.2% lysine in
0.1M phosphate buffer.
4% paraformaldehyde with
0.05% glutaraldehyde
10% Neutral Buffered Formalin

11. Antigen Retrieval
Heat Induced Epitope Retrieval
➔ Unmask hidden antigenic sites
➔ Break protein crosslinks formed by formalin fixation
Proteolytic Induced Epitope
Retrieval

12. Applications of IHC
Identifying cancer
cells
Differentiate between benign and
malignant cancer
Identifying
secondary tumor
deposit.
It can be used in hospitals, research labs, pharmaceutical industries
and educational institution.

13. Advantages of IHC
➔ Wide range of specific antibodies
➔ Highly sensitive detection system.

14. Thank you!