Using Antibodies in Microscopy: A Guide to Immunohistochemistry. Part 3: Post-Embedding Electron Microscopy Techniques

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. In practice, however, an IHC protocol has many steps where problems can occur resulting in either false positive or false negative data. IHC can be used for ultrastructural studies using electron microscopy (EM), but the protocols used for light microscopy must be adapted. While IHC techniques have been published extensively, a certain amount of confusion exists as to how to optimize them. This series of articles describes the steps involved in IHC and 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.