Simultaneous ultrastructural demonstration of multiple peptides in endocrine cells by a novel immunocytochemical method

Abstract

Current immunocytochemical techniques detect all antibodies that react with tissue. Unfortunately, some of these antibodies may react with antigens other than those intended. Such problems are minimised by using sets of antibodies detecting different regions of the desired antigen. However, as immunocytochemical methods can now detect very low antibody concentrations, the purity of antisera is critical. Furthermore, although antisers may be purified by affinity chromatography, difficulties in recovering high-avidity antibodies cause most affinity-purified antisera to be enriched in low-avidity antibodies, which may be dislodged during staining. We have therefore developed, and describe here, a new ultrastructural post-embedding staining technique, based on the divalency of IgG molecules and using antigen-coated colloidal gold granules. Previously, colloidal gold-labelled antibodies have been used for post-embedding staining. Unlike our gold-labelled antigen detection (GLAD) technique, however, these methods do not differentiate between specific and nonspecific antibodies. The GLAD method detects only specific antibodies and does not select against high-avidity antibodies, and in this it resembles the radioimmunocytochemical method. However, the GLAD method differs from the latter in that it is useful for ultrastructural studies, does not require autoradiography and allows simultaneous detection of multiple antigens. Moreover, specific activity compared with background may be quantitated.