Using Antibodies in Microscopy: A Guide to Immunohistochemistry. Part 2: IHC Staining Protocols

Abstract Immunohistochemistry (IHC) is the use of antibodies as probes to determine the localization of proteins in a cell or tissue sample. In microscopy, the technique has been in use since the 1940s by pathologists and research scientists. At first glance it appears a straightforward procedure. However, in practice, an IHC protocol has a number of steps where problems can occur, resulting in either false positive or false negative data. IHC can be used in both transmitted light and fluorescence microscopy, and it can be used in combinations to identify and mark multiple proteins of interest. While the technique has been published extensively, a certain amount of confusion exists as to how to optimize it. This series of three articles describes the steps involved in IHC, their function, and some variations that occur in specific protocols on differing cell and tissue types. It is hoped that a better understanding of these steps will help to guide users in determining the optimal conditions for carrying through IHC protocols.