AbstractThis unit describes several methods for localizing specific antigens in various tissue and cell preparations using immunohistochemistry (IHC). Protocols describe preparation of suitable material for IHC including fresh, unfixed, frozen tissue specimens; unfixed cells, either freshly isolated or from suspension or adherent cultures, or fixed, paraffin‐embedded tissue sections. By careful selection of reagents, it is possible to detect two antigens simultaneously. For antigens that are sensitive to fixative, it may be necessary to unmask the antigen by a new technique called “antigen retrieval”. If there is cross‐reactivity between the secondary antibody and antigens present in the target cells or tissue, the secondary antibody can be preabsorbed. The different IHC protocols are represented schematically and summarized in a table, which also lists advantages and disadvantages of each approach. Causes of background staining and ways to eliminate it are also discussed.