INTRODUCTIONLike RNA, proteins can be localized in situ on tissue sections. In situ localization or immunohistochemistry provides information on the cellular and subcellular distribution of proteins in different tissues. The technique described in this protocol employs a biotinylated secondary antibody in conjunction with a preformed Avidin:Biotinylated enzyme Complex (ABC) for immunohistochemistry. It allows for sensitive detection of antigens while avoiding fluorescent secondary antibodies, which produce a much less stable signal. Formation of the complex is achieved by mixing avidin and biotinylated enzyme in dilute solution and in defined amounts prior to use. The complex is then allowed to bind to biotinylated secondary antibodies, thus linking the antigen via primary and secondary antibodies to the enzyme used to generate the signal. Kits for this procedure are available from Vector Laboratories. The VECTASTAIN ABC kits are of three types, using the enzymes horseradish peroxidase, alkaline phosphatase, or glucose oxidase. This protocol assumes use of the peroxidase kit, but can be easily modified for the other kits.