/  VECTASTAIN ABC Systems   How do you use the "ABC Method"?
      

The first step of the procedure is to incubate the section with primary antiserum raised against the antigen of interest, such as rabbit antibody to a tumor-associated antigen. Next, a biotin-labeled secondary antibody is added, which in this example would be biotinylated anti-rabbit IgG. This introduces many biotins into the section at the location of the primary antibody. The avidin : biotinylated enzyme complex (ABC) is then added and binds to the biotinylated secondary antibody. In the last step of the procedure, the tissue antigen is localized by incubation with a substrate for the enzyme.

In some cases it may be necessary to precede these steps with blocking procedures. To eliminate any nonspecific adsorption of the primary antibody to tissue elements, dilute serum from the same species as the biotinylated secondary is added as a first step. If the tissue contains endogenous peroxidase (which will produce a reaction product from the substrate alone), a variety of enzyme blocking procedures can be employed. If alkaline phosphatase activity is present in the tissue section, levamisole can be added to the substrate solution. Some tissues may contain endogenous biotinylated proteins. If this occurs, an avidin/biotin blocking step can be used. In general, the procedure is easy to use and will give the highest quality staining of any immunohistochemical method.