Manual Immunohistochemistry Staining of Mouse Tissues Using the Avidin–Biotin Complex (ABC) Technique

Abstract

There are many variations on the immunohistochemistry (IHC) procedure, but all are based on attachment of a primary antibody to a unique epitope on or within the cell. This step is followed by incubation of the cell/primary antibody complex with another, secondary antibody that recognizes the species in which the primary antibody was produced. The secondary antibody has an indicator molecule attached to it. The indicator produces a colored reaction product at the site of original epitope, allowing visualization. This basic two-antibody “sandwich” procedure has many modifications that include other layers of antibodies and numerous indicators, but all variations depend upon the unique ability of antibodies to recognize specific epitopes or antigenic determinants. The procedure described here is called the ABC (avidin–biotin complex) technique. The method utilizes the high avidity of biotin for avidin, which allows formation of a strong bond. The reagents described in this technique produce a gold/brown reaction product that identifies the epitope of interest.