Immunohistochemistry

1. Immunohistochemistry

2. Introduction
• Immunohistochemistry (IHC) combines histological,
immunological and biochemical techniques for the
identification of specific tissue components by means of
a specific antigen/antibody reaction tagged with a visible
label.
• IHC makes it possible to visualize the distribution and
localization of specific cellular components within a cell
or tissue.

3. • Application of antibodies to tissue preparation for
the localization of target antigens:
• Wide range of specific antibodies
• Highly sensitive detection system

4.  Immunohistochemistry utilizes labeled antibodies to
localize specific cell and tissue antigens, and is among the
most sensitive and specific histochemical techniques.
 Because many targeted antigens are proteins whose
structure might be altered by fixation and clearing, so
frozen sections are commonly used.
 In some cases, paraffin wax can be used for embedding.

5. Immunohistochemistry assays may use
Cells grown, spun into a pellet, frozen or
paraffin embedded and sectioned
Cells grown as a monolayer
OR use tissue sections that are frozen or paraffin embedded
Sections from tissues contain many different kinds of cells
as well as extra-cellular matrix components
cells on slides

6. If the tissue is frozen The sections may need to be used
in immunohisto-assays as
Tissue section on glass slide: Frozen
Acetone fixed:
- precipitates proteins onto cell surface---may extract lipids
-is needed for many of the “CD” antibodies
Unfixed:
Positive feature:-antigens are unaltered
Negative feature: sections may fall off slide during staining
Paraformaldehyde fixed:
--needs to be freshly made, or frozen soon after
--is preferred over using 10% buffered formalin

7. Tissue section: Paraffin embedded
If the tissue is paraffin embedded,
- Deparaffinize ( remove the infiltrated paraffin wax,
by using organic solvents)
- The section then needs to be rehydrated, by sequential immersion
in graded alcohols (100%, 70% , 50% and then PBS)
- The deparaffinized section may need to be treated
to expose buried antigenic epitopes
with either proteases
or by heating in low pH citrate buffer ,
or high pH EDTA buffer (Antigen Retrieval)

8. Principle
• The principle of immunohistochemistry is the
localization of antigens in tissue sections by the use of
labeled antibodies as specific reagents through antigen-
antibody interactions that are visualized by a marker
such as fluorescent dye, enzyme, radioactive element or
colloidal gold.

9. Antibodies (Immunoglobulins)
• Glycoprotein that are produced by plasma
cells and used by the immune system to
identify and neutralise foreign objects, ie.
bacteria and viruses
• recognise a specific Antigen- mainly
proteins, glycoprotein, polysaccharides
• Complementary Determining Region

10. Antigen Detection

11. Antibodies binding to Antigens

12. Antigens

13. A. Raising Antibodies:
• Repeated injection of antigens (proteins, glycoproteins,
proteoglycans, and some polysaccharides) causes the
injected animal's B lymphocytes to differentiate into
plasma cells and produce antibodies.
• Members of a lymphocyte clone (descendents of a single
lymphocyte) produce a single type of antibody, which
binds to a specific antigenic site, or epitope.

14. 1. Polyclonal antibodies: Large complex antigens may
have multiple epitopes and elicit several antibody types.
Mixtures of different antibodies to a single antigen are
called polyclonal antibodies.
2. Monoclonal antibodies: Antibodies specific for a single
epitope and produced by a single clone are called
monoclonal antibodies and are commonly raised in mice.

15. B. Labeling Antibodies:
• Antibodies are not visible with standard microscopy and
must be labeled in a manner that does not interfere with
their binding specificity.
• Common labels include fluorochromes (eg, fluorescein,
rhodamine), enzymes demonstrable via enzyme
histochemical techniques (eg, peroxidase, alkaline
phosphatase), and electron-scattering compounds for use
in electron microscopy (eg, ferritin, colloidal gold).

16. Method
• Direct Method
• Indirect Method
• PAP Method

17. Direct Method
Tissue Antigen
Labeled Antibody

18. Two-Step Indirect Method
Tissue Antigen
Primary Antibody
Secondary Antibody

19. PAP Method
(peroxidase anti-peroxidase method)

20. Applications
• Cancer diagnostics
• differential diagnosis
• Treatment of cancer
• Research

21. General Immunohistochemistry
Protocol

22. Part 1
1. Fixation
Fresh unfixed, fixed, or formalin fixation and
paraffin embedding
2. Sectioning
3. Whole Mount Preparation
Tissue preparation

23. Part 2
1. Antigen retrieval
Proteolytic enzyme method and Heat-induced method
2. Inhibition of endogenous tissue components
3% H2O2, 0.01% avidin
3. Blocking of nonspecific sites
10% normal serum
pretreatment

24. Part 3
• Make a selection based on the type of
specimen, the primary antibody, the degree
of sensitivity and the processing time required.
staining

25. Controls
• Positive Control
It is to test for a protocol or procedure used.
It will be ideal to use the tissue of known positive as a
control.
• Negative Control
It is to test for the specificity of the antibody involved.