Ultrastructural immunochemistry

INTRODUCTIONThe use of colloidal gold technology was undoubtedly the most significant event in the development of immunochemistry. Gold particles are particularly useful for transmission electron microscopy (TEM) studies, because they scatter electrons strongly and even small particles are clearly visible under the electron microscope. Before proceeding to immunogold staining, it is important to gather as much information as possible about the antibody of interest and its respective antigen: Where is it likely to be located? Is the antigen extracellular, intracellular, membrane-associated, or a soluble component of the cytoplasm? Is it present in significant quantities? Is it sequestered at high concentration in any specific subcellular compartment, such as the mitochondria or the nucleus? How vulnerable to fixation and embedding is the antigen of interest? Information on the specificity of antibodies from Western blotting is valuable, but is not guaranteed to be useful for immunochemistry. Antibodies that "work well" on blots frequently have to be used at concentrations of up to three or more orders of magnitude greater for immunofluorescence and even more for immunogold staining studies, and some antibodies simply cannot be used for immunochemistry. This article describes methods and considerations for the use of immunogold staining, including fixation, controls, resolution, and quantification.