1. Practical of Histopathology
بسم الله الرحمن الرحيم
Lab 6 Immunohistochemistry (IHC)
Practical of Histopathology

2. Overview Immune System
Molecules, cells, tissues and organs which provide protection against (Microorganisms, Microbial toxins, Tumor cells).
Antigens
are substances that induce a specific immune response and subsequently react with the products of a specific immune response.
Antibodies (Immunoglobulin)
are a type of glycoprotein produced by B lymphocytes.

3. Epitope, or , Antigenic determinants
Are the portions of antigen molecules that physically interact with paratopes.
Paratopes.
Are the(combining sites) of immune response molecules and therefore actually "determine" antigen specificity.
Immunohistochemistry (IHC)
It 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.

4. The body's response to the introduction of a foreign agent, known as the immune response, results in the production of antibodies which bind the offending material.
Antibodies bind tightly and specifically to an "epitope" (one specific structure) on an "antigen" (foreign molecule or structure).

5. Fab region
Paratope + Epitope
Ab-Ag complex

6. Types of antibodies
Polyclonal
Ag injected into host animal. Serum collected and purified.
Multiple antibodies produced by different cell types bind multiple epitopes on Ag.
2. Monoclonal
Ag injected into mouse. Lymphocytes isolated, hybridized.
One antibody produced by one cell type binds one epitope on Ag.

7. Not all antigens directly elicit an antibody response.
Some require a carrier to be effective. These generally smaller antigens are called haptens.
Haptens are small molecules that elicit an immune response only when attached to a large carrier such as a protein; the carrier may be one that also does not elicit an immune response by itself.

8. Antibodies can be generated by injecting animals with antigens, and then collecting serum after the immune response has taken place.
If the antibodies are labeled with an easily detectable molecule (a fluorescent dye, an enzyme, etc.), they become powerful detection reagents for the antigen.
This system has been exploited to generate exceptionally specific and sensitive "stains" which are used in histology as well as other disciplines.

9. The basic process depends upon selecting an antibody sufficiently specific to bind an antigen in situ.
The antibody/antigen conjugate is then identified using a variety of signal generating molecules triggered either by the antibody/antigen interaction or by secondary processes.
The signal generators can be precipitating dyes, fluorescent molecules or electron dense (ultrastructure tag) materials for electron microscopy (EM).

11. Immunohistochemistry is generally carried out in:
sectioned tissue, which allows the antibodies free access to the interior of the cells.
cells either in free solution or bound to membranes, or on monolayers of cultured cells.
Intracellular Immunohistochemistry requires that:
the antibody to the target antigen be able to penetrate the cell membrane. whatever cell wall may be present before it can attach to the antigen.

12. This requires a number of steps not required for sectioned tissue.
Primarily the cell membrane must be made permeable to the antibody, though at the same time the integrity of the cell contents and structures must be maintained.
This is normally achieved through the use of a specialized buffer containing a detergent.

14. Methods of antibody labeling
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.

15. 2. 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.

16. The indirect method is more sensitive than the direct method because:
numerous labeled anti-antibodies bind to the primary antibody, making them easier to visualize.
the indirect method does not require labeling of the primary antibody, which often is available only in limited quantities.
Immunocytochemistry can be used with specimens for electron microscopy by labeling the antibody with ferritin, an electron- dense molecule, instead of with a fluorescent dye.
Ferritin labeling can be applied in both the direct and indirect methods.

17. General Immunohistochemistry Protocol
Part 1 Tissue preparation 1. Fixation (Fresh unfixed, fixed, or formalin fixation and paraffin embedding). 2. Sectioning. 3. Whole Mount Preparation. Part 2 pretreatment 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).

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

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

20. Uses of immunohistochemistry
1- Locate cells that are signaling. 2- Identify replicating cells. 3- Locate apoptotic cells. 4- Identify activation states. 5- Identify different types of cells in a tissue. 6- Examine cytoskeletal structure.