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 derived from suspension or adherent cultures; or fixed, paraffin‐embedded tissue sections. By careful selection of reagents, it is possible to detect two or even three antigens simultaneously. For antigens that are sensitive to fixative, it may be necessary to unmask the antigen by the antigen‐retrieval technique. If there is cross‐reactivity between the secondary antibody and antigens present in the target cells or tissue, the secondary antibody can be preabsorbed. Several new, sensitive amplification techniques are currently available. The different IHC protocols are represented schematically and summarized in a table that also lists advantages and disadvantages of each approach. Causes of background staining and ways to eliminate it are also discussed. Curr. Protoc. Immunol. 103:21.4.1‐21.4.26. ©2013 by John Wiley & Sons, Inc.