Localization of the myc-Tagged 5HT_5A Receptor by Immunogold Staining of Ultrathin Sections

Abstract

Immunohistochemistry is a powerful technique to localize proteins in tissues and cultured cells as well as in fractions of subcellular compartments, like mitochondria, endoplasmic reticulum, vesicles, and membranes. In most cases, the detection of a specific protein occurs by a sandwich approach, consisting of a specific primary antibody directed against the target protein and a secondary antibody directed against the primary antibody, which is coupled to a marker for subsequent visualization. Therefore, a key prerequisite for the successful localization of a target protein is the availability of a high-quality specific primary antibody preparation. The production of specific polyclonal antibodies or monoclonal antibodies (MAb) can be time-consuming and cumbersome, and must be performed for each new target protein. Furthermore, membrane proteins are often poorly antigenic. Additional difficulties may arise when an antibody preparation raised against the target protein has a high background of nonspecific antibodies or when the antibodies, despite working well for Western blot analysis, do not work well for immunohistochemical studies.