The Basics Technique of Immunohistochemistry

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     1  By Sri Widyarti ( Dept. of Biology, Brawijaya University The Basics Technique of Immunohistochemistry
     2  Definitions Immunohistochemistry is a technique for identifying tissue constituents (antigens) by means of antigen-antibody interactions. The site of antibody binding being identified either by direct labeling of the antibody, or by use of secondary antibody labeling method.
     4  Steps of Immunohistochemistry Tissue sections are incubated with primary antibodies against the target antigen. The specimens are then washed and a secondary antibody against primary antibody is added. The secondary antibodies are often biotinylated, that is, conjugated to biotin, a vitamin with an extremely high affinity for the protein streptavidine.
     5  A complex made of streptavidin and the enzyme peroxidase is used for staining. The addition of a chromogenic substrate such as diaminobenzidine (DAB) or aminoethylcarbazole (AEC), leads to a color staining reaction that accurately reflects the distribution of the target antigen in the tissue sample.
     6  Unmasking of Antigen Sites Tissue fixation on routinely histotechnique can masked antigens because of formalin cross-linking or even destroys some antigenic epitopes. The retrieval techniques of unmasked antigens : (1) proteolytic enzyme digestion (2) microwave (3) microwave and trypsin (4) pressure cooker
     7  Antigen Recognition 1’Ab : primary antibody 2’Ab : secondary antibody B : biotin SA : streptavidine HRP : horseradish peroxidase Target is antigen (protein argets) Blocking reagent : BSA (bovine serum albumin) The sandwich of antigen recognition on tissue samples.
     8  The methods of Identifying Tissue Antigens Direct labeling of antibody Secondary antibody labeling
     9  Labels Enzyme labels Colloidal metal labels Fluorescent labels Radiolabels
     10  Enzyme Labels Horseradish peroxidase Alkaline phosphatase Bacterial-derived beta-D-galactosidase
     11  Several alternative chromogens for demonstration of peroxidase
     12  Properties of Fluorochrome
     13  Colloidal metal labels Immunogold method
     14  Several alternative chromogens for demonstration of others labeled enzyme
     15  The microscopy Observation of Immunohistochemistry Immunohistochemstry of lungs benzo(a)pyrene-injected rat which stained anti-PARP, secondary antibody HRP-labeled, and AEC chromogenic substrate to visualization (Widyarti, 2005).
     20  End