Introduction to Immunohistochemistry

Study Notes

Immunohistochemistry (IHC) is a technique used to identify and localize specific antigens in tissue sections.
IHC relies on the specific binding of antibodies to their corresponding antigens.
The antigen-antibody interactions are visualized using markers, such as fluorescent dyes, enzymes, radioactive elements, or colloidal gold.
IHC was first used in 1941 by Albert H Coons and colleagues.

Cellular antigens bind to added antibodies, forming an immune complex.
The immune complex is labeled with an enzyme or fluorescent dye, making it soluble and colored.
The colored substance is reacted with a chromogen (e.g., diaminobenzidine) to produce an insoluble, colored substance that can be visualized.

The process involves tissue fixation and processing, creating 3-5 micron thick sections on coated slides.
Deparaffinization (dewaxing) and rehydration are applied to the tissue sections.
Antigen retrieval techniques, either heat-induced epitope retrieval (HIER) or proteolytic induced epitope retrieval (PIER), are performed to expose hidden antigenic sites.
Endogenous peroxidase blocking is necessary to prevent nonspecific background staining.
The primary antibody is applied, followed by the secondary antibody.
The substrate and chromogen are selected based on the method used, leading to visualization of the target antigen.

Immunoenzyme Histochemistry: Uses enzymes attached to antibodies.
- Commonly used enzymes are Horseradish Peroxidase (HRP) or Alkaline Phosphatase.
- Visualization is achieved using a light microscope.
- Most commonly used IHC method.
Immunofluorescence Histochemistry: Uses fluorescent dyes attached to antibodies.
- Fluorophores like FITC, Texas Red, Rhodamine, or DyLight Fluor are used.
- Visualization is achieved using a fluorescent microscope.

Direct Method: The primary antibody directly binds to the target antigen and is labeled with an enzyme.
Indirect Method: Uses a secondary antibody labeled with an enzyme that recognizes the primary antibody, providing signal amplification.
PAP (Peroxidase Anti-Peroxidase) Method: Employs an antibody directed against the peroxidase enzyme, leading to enhanced signal amplification.
ABC (Avidin-Biotin Complex) Method: Utilizes biotinylated secondary antibodies and avidin-biotin complexes to amplify the signal.
LSAB (Labeled Streptavidin Biotin) Method: Uses a streptavidin molecule conjugated to an enzyme, creating a strong signal.
Polymeric Methods: Employ polymers conjugated with enzymes and antibodies for more efficient signal amplification.
- Examples include the EnVision System and the ImmPRESS System.
CSA (Catalyzed Signal Amplification) Methods: Utilize tyramide-based amplification techniques for highly sensitive detection.
- CSA I uses biotinyl-tyramide, while CSA II uses fluorescyl-tyramide.

This process is crucial to unmask hidden antigenic sites that can be masked by formaldehyde fixation.
Two main techniques are used for antigen retrieval:
- Heat-Induced Epitope Retrieval (HIER): Tissue sections are heated using devices like microwave ovens, pressure cookers, or steamers.
- Proteolytic Induced Epitope Retrieval (PIER): Enzymes such as proteinase K, trypsin, chymotrypsin, pepsin, pronase are used to digest protein cross-links.

Some cells and tissues contain endogenous peroxidase, which can cause nonspecific background staining when using HRP-conjugated antibodies.
Pre-treatment with 0.3% to 3.0% hydrogen peroxide before HRP antibody incubation helps reduce this nonspecific background.

The choice of method depends on the specific IHC method used. All methods involve the use of primary and secondary antibodies.
- Direct Method: Primary antibody directly binds to target antigen.
- Indirect Method: Secondary antibody recognizes the primary antibody.
- PAP, ABC, LSAB, Polymeric, CSA: All these methods rely on secondary antibodies and various signal amplification techniques.

Cytokeratin: A marker for carcinomas (cancers of epithelial origin).
LCA (Leukocyte Common Antigen): Marker for leukemias (cancers of the blood-forming tissues).
CD15 & CD30: Found in Hodgkin's lymphoma.
AFP (Alpha-fetoprotein): Marker for hepatocellular carcinoma (liver cancer).
CD117: Marker for Gastrointestinal Stromal Tumors (GIST).
PSA (Prostate-Specific Antigen): Marker for prostate cancer.
Desmin: Marker for non-epithelial tumors (e.g., muscle tumors).
CD20: Marker for B-cell lymphomas.
CD3: Marker for T-cell lymphomas.
GFAP (Glial Fibrillary Acidic Protein): Marker for brain tumors.
Myosin: Marker for muscle tumors.
S100 Protein: Marker for melanomas (skin cancers).