Immunolabeling for electron microscopy

The protocols in this chapter concern postembedding immunolabeling for transmission electron microscopy; other schedules, such as pre-embedding methods, frozen tissue processes, and procedures for scanning electron microscopy, can be found elsewhere (1). In principle, immunolabeling at the electron microscope (EM) level follows the same precepts as immunolabeling at the light microscope level; in tissues or cells, the location of an antigen of interest is identified by a specific antibody, and must be visualized appropriately for investigation. Electron microscopy permits us to distinguish subcellu-lar organelles, and therefore ultrastructural localization of antigen position. At the EM level, however, the "visualizing step" needs to be provided by an electron-dense entity, most often a heavy metal, which reflects incident electrons; this is in contrast to the final step of light microscope level techniques, in which the final reaction product is sought to be colored (and where there is an element of choice of which color to use). Tissue processing for EM is considerably more severe than that for light microscopy, and thus maintenance of antigenicity in tissue is more taxing. Before immunolabeling for electron microscopy can be fruitful, the first step is to ensure that the antigen of interest is present (or is still present) in the tissue; this is done by performing a thorough procedure at the light microscope level on wax-embedded sections. Once a positive result has been obtained, studies can progress to the ultrastructural level. If the presence of an antigen cannot be demonstrated in a wax-embedded block, it will not be demonstrable in a resin-embedded EM block of the same tissue. In such a case, pre-embedding and frozen tissue techniques can be of use at both light and electron microscope levels.