Immunohistochemistry (IHC) combines histology and immunology to identify specific tissue components using antigen-antibody reactions tagged with visible labels. The principle involves localizing antigens in tissue sections using labeled antibodies. Antibodies are raised against antigens and labeled with fluorescent dyes, enzymes, or metals. IHC methods include direct, indirect, and peroxidase anti-peroxidase. IHC has applications in identifying cell types, markers for cancer prognosis and therapy response, detecting infections, and examining cytoskeletal structure. The general IHC protocol involves tissue fixation, antigen retrieval, blocking, primary/secondary antibody incubation, and controls.

1. Immunohistochemistry (IHC)
By PhD Student
Haael S. Abbas
Supervised By
Dr. Hind J. Hassoon

2. Introduction
• Imunohistochemistry
(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.
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3. • IHC is an application of antibodies to tissue
preparation for distribution and localization of
target antigens:
• Wide range of specific antibodies
• Highly sensitive detection system

4. Principle
• The principle of immunohistochemistry is to localize
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.

5. 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.

6. 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.
ANTIBODIES (IMMUNOGLOBULINS)

7. 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).

8. Method
• Direct Method
• Indirect Method
• PAP Method

9. Direct method
• The antibody against the
macromolecule is labeled with a
fluorescent dye.
• The antibody is then permitted to
react with the macromolecule, and
the resultant complex may be
viewed with a fluorescent
microscope.

10. Indirect method
•a fluorescent labeled antibody is
prepared against the primary
antibody specific for the
macromolecule of interest.
•forming a secondary complex
visible by fluorescent microscopy

12. PAP Method
(peroxidase anti-peroxidase method)

13. Applications
• Identify replicating cells
• Locate cells that are signaling
• Locate apoptotic cells
• Identify activation states
• Identify different types of cells in a tissue
• Examine cytoskeletal structure

14. Applications
1. Tumors of uncertain histogenesis. IHC has brought about a
revolution in approach to diagnosis of tumors of uncertain
origin, primary as well as metastatic from an unknown
primary tumor.
2. Prognostic markers in cancer. IHC is to predict the prognosis
of tumors by detection of micrometastasis, occult
metastasis, and by identification of certain features
acquired, or products elaborated, or genes overexpressed,
by the malignant cells to predict the biologic behavior of
the tumor. These include: proto-oncogenes (e.g. HER-2/neu
overexpression in carcinoma breast), tumor suppressor
genes (e.g. Rb gene, p53), growth factor receptors (e.g.
epidermal growth factor receptor or EGFR).

15. Applications
3. Prediction of response to therapy. IHC is widely used to
predict therapeutic response in two important tumors—
carcinoma of the breast and prostate. Both these tumours
are under the growth regulation of hormones—oestrogen
and androgen, respectively. The specific receptors for
these growth regulating hormones are located on
respective tumor cells.
4. Infections. IHC stains are now being applied to confirm
infectious agent in tissues by use of specific antibodies
against microbial DNA or RNA e.g. detection of viruses
(HBV, CMV, HPV, herpesviruses), bacteria (e.g. Helicobacter
pylori), and parasites (Pneumocystis carinii ) etc.

17. General Immunohistochemistry
Protocol

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

19. Part 2
1. Antigen retrieval (The deparaffinized section may
need to be treated to expose buried antigenic epitopes)
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

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

21. • Positive Control
It is to test for sensitivity of the antibody involved.
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.
Controls

22. What cellular antigens
can we target?
• Cytoplasmic
• Nuclear
• Cell membrane
• Lipids
• Proteins
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