Immunohistochemistry Lecture Notes PDF

Immunohistochemistry 231 MLT Histology  Definition  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 vi...

Immunohistochemistry 231 MLT Histology  Definition  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  IHC Principle  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 IHC Methods  Direct Method  Indirect Method PAP /APAAP Method ABC Method SP Method  IHC Methods Wide range of IHC methods is available, so make selection based on: Type of specimen Primary antibody Degree of sensitivity and the processing Time required Cost of reagents  Direct Method Labeled Antibody Tissue Antigen  Two-Step Indirect Method Secondary Antibody Primary Antibody Tissue Antigen  PAP Method (peroxidase anti-peroxidase method)  Avidin-Biotin Complex (ABC) Method The ABC method incorporates a large glycoprotein called avidin that has a very high affinity for a low molecular weight vitamin called biotin. Avidin can be labeled with peroxidase or fluorescein. Biotin can be conjugated to antibodies. The technique methodology consists of using an unlabeled primary antibody, a biotinylated secondary antibody, and a complex of avidin-biotin peroxidase. The peroxidase is then developed by the DAB or other substrates to produce a colored label. Streptavidin Peroxidase Conjugated Method (SP Method)  SP Method (streptavidin peroxidase conjugated method)  Applications  Cancer diagnostics  differential diagnosis  Treatment of cancer  Research General Immunohistochemistry Protocol  Protocol overview 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  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  Part 1 Tissue preparation 1. Fixation - formalin fixation and paraffin embedding 2. Tissue processing 2. Tissue sectioning 3. Whole Mount Preparation  Part 2 pretreatment 1. Antigen retrieval a. Heat-induced method b. Proteolytic enzyme method 2. Inhibition of endogenous tissue components 3% H2O2 or 0.01% avidin 3. Blocking of nonspecific sites 10% normal serum  Part 3 staining  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  Part 3 staining  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 often applied after the chromogen  Troubleshooting A. Weak or No Staining B. Over-staining C. High Background  Weak or No Staining 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  Over-staining 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  High Background 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