Immunohistochemistry Lecture Notes PDF

Immunohistochemistry (IHC) is combined 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 visualize the distribution and localization of specific cellular compon...

Immunohistochemistry (IHC) is combined 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 visualize the distribution and localization of specific cellular components within a cell or tissue The principle of IHC is the localization of antigens in tissue sections by the use of labeled antibodies through antigen- antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold - **Direct Method** - **Indirect Method** - **PAP /APAAP Method** - **ABC Method** - **SP Method** - Type of specimen - Primary antibody - Degree of sensitivity and the processing - Time required - Cost of reagents **Secondary Antibody**  Avidin-Biotin Complex (ABC) Method ==================================  - Cancer diagnostics - differential diagnosis - Treatment of cancer - Research  1. Prepare and fix tissue 2. Antigen retrieval 3. Suppress endogenous peroxidase activity 4. Block nonspecific sites in the tissues 5. Incubate the tissues with the primary antibody 6. Incubate the tissues with the secondary antibody 7. Incubate the tissues with SP 8. Add DAB and incubate until desired staining is achieved - **Positive Control** - **Negative Control** 1. **Fixation -** formalin fixation and paraffin embedding 2. **Tissue processing** 2. **Tissue sectioning** 3. **Whole Mount Preparation** 1. Antigen retrieval ----------------- a. Heat-induced method b. Proteolytic enzyme method 2. Inhibition of endogenous tissue components ------------------------------------------ Blocking of nonspecific sites ----------------------------- - Target Antigen - Proteins that are within or on the surface of a cell - Primary antibody - Two main types of antibody, polyclonal and monoclonal - Secondary antibody - Bind to the primary antibody - Detection system - Builds on the secondary antibody - Chromogenic detection utilizes enzymes such as Horseradish Peroxidase (HRP) that are conjugated to antibody - The chromogen - Substrate forms an insoluble colored precipitate that can be visualized under a microscope - Two commonly used chromogens: - 3,3\'-Diaminobenzidine (DAB) stain brown color - Alkaline phosphatase (AP) stain with red color - DAB used most application - AP used mainly for skin sections - - The blue background is a hematoxylin counter-stain that is A. Weak or No Staining B. Over-staining C. High Background 1. Inadequate deparaffinization 2. Inactive primary antibodies 3. Antibodies do not work due to improper storage 4. Antibody concentration was too low 5. Inadequate antibody incubation time 6. Inadequate or improper tissue fixation 7. Tissue over fixation 8. Incompatible secondary and primary antibodies 9. Inactive secondary antibody or other reagents 10. Inadequate substrate incubation time 11. Reagents applied in wrong order or steps omitted 12. Incorrect mounting medium 1. The concentration of antibodies was too high 2. Incubation time was too long 3. Incubation temperature was too high 4. Substrate incubation time was too long 5. Sections dried out 1. Inadequate washing of sections 2. Tissue contains endogenous enzyme 3. Tissue contains endogenous biotin activity 4. Non-specific binding of primary antibodies to tissue or high antibody concentration 5. Non-specific binding of secondary antibodies to tissue 6. Diffusion of tissue antigen due to inadequate fixation