1. King Saud University Riyadh Saudi Arabia
Dr. Gihan Gawish
Assistant Professor
Dr. Gihan Gawish

2. Immunological Methods
Immunohistochemistry
Dr. Gihan Gawish

3. Immunohistochemistry )IHC)
flowcytometry cont'd
Immunohistochemistry )IHC)
refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues
It takes its name from the roots "immuno," in reference to antibodies used in the procedure, and "histo," meaning tissue
Dr. Gihan Gawish

4. Immunohistochemical staining
Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors.
Specific molecular markers are characteristic of particular cellular events such as proliferation or cell death (apoptosis)
. IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue.
Dr. Gihan Gawish

5. Visualising an antibody-antigen interaction can be accomplished in a number of ways.
In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase that can catalyze a color-producing reaction
Alternatively, the antibody can also be tagged to a fluorophore, such as FITC
FITC is a derivative of fluorescein used in wide-ranging applications including flow cytometry).
Dr. Gihan Gawish

6. Immunophenotyping
Immunophenotyping is a technique used to study the protein expressed by cells.
This technique is commonly used in basic science research and laboratory diagnostic purpose.
This can be done on tissue section (fresh or fixed tissue), cell suspension, etc.
Dr. Gihan Gawish

7. Immunophenotyping An example is the diagnosis of leukemia.
It involves the labeling of white blood cells with antibodies directed against surface proteins on their membrane.
By choosing appropriate antibodies, the differentiation of leukemic cells can be accurately determined.
Dr. Gihan Gawish

8. Immunophenotyping
The labeled cells are processed in a flow cytometer, a laser-based instrument capable of analyzing thousands of cells per second.
The whole procedure can be performed on cells from the blood, bone marrow or spinal fluid in a matter of a few hours.
An Example of Information provided through Immnunophenotyping "The flow cytometric immunophenotyping report indicated the malignant cells were positive for CD19, CD10, dimCD20, CD45, HLA-DR, and λ immunoglobulin light chain. There was no coexpression of CD5 or CD23 by the monoclonal B-cell population."
Dr. Gihan Gawish

9. Antibody types
The antibodies used for specific detection can be polyclonal or monoclonal.
Monoclonal antibodies are generally considered to exhibit greater specificity.
Polyclonal antibodies are made by injecting animals with peptide antigens, and then after a secondary immune response is stimulated, isolating antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix of antibodies that recognize several epitopes.
Dr. Gihan Gawish

10. Antibody types
Antibodies can also be classified as primary or secondary reagents.
Primary antibodies are raised against an antigen of interest and are typically unconjugated (unlabelled),
while secondary antibodies are raised against primary antibodies.
Hence, secondary antibodies recognize immunoglobulins of a particular species and are conjugated to either biotin or a reporter enzyme such as alkaline phosphatase or horseradish peroxidase (HRP).
Some secondary antibodies are conjugated to fluorescent agents
Dr. Gihan Gawish

11. Sample preparation
In the procedure, depending on the purpose and the thickness of the experimental sample, either thin (about 4-40 μm) slices are taken of the tissue of interest, or if the tissue is not very thick and is penetrable it is used whole.
The slicing is usually accomplished through the use of a microtome, and slices are mounted on slides.
Dr. Gihan Gawish

12. Immunohistochemical methods
There are two strategies used for the immunohistochemical detection of antigens in tissue the direct method and the indirect method.
In both cases, the tissue is treated to rupture the membranes, usually by using a kind of detergent such as Triton X-100.
Dr. Gihan Gawish

13. Direct Method
The direct method is a one-step staining method, and involves a labeled antibody (e.g. FITC conjugated antiserum) reacting directly with the antigen in tissue sections.
This technique utilizes only one antibody and the procedure is therefore simple and rapid.
However, it can suffer problems with sensitivity due to little signal amplification and is in less common use than indirect methods.
Dr. Gihan Gawish

14. Indirect Method
The indirect method involves an unlabeled primary antibody (first layer) which reacts with tissue antigen, and a labeled secondary antibody (second layer) which reacts with the primary antibody.
(The secondary antibody must be against the IgG of the animal species in which the primary antibody has been raised.) This method is more sensitive due to signal amplification through several secondary antibody reactions with different antigenic sites on the primary antibody. The second layer antibody can be labeled with a fluorescent dye or an enzyme
Dr. Gihan Gawish

15. See the Immunohisochemistry animation in my site
Dear my students,
See the Immunohisochemistry animation in my site
Dr. Gihan Gawish