Immunocytochemical localization of intracellular antigens with SEM

A method for the localization of intracellular antigens with a scanning electron microscope using peroxidase-labelling antibodies is described. A search for a hydrogen donor which may be deposited at the sites of antigen by enzymatic action and emit secondary electrons or generate backscatter electrons was made. It was found that when 4-chloro-1-naphthol was used, the peroxidase deposited reaction product which resulted in a strong secondary electron emission at the site of antigen. With this method, the presence of luteinizing hormone in secretion granules and other cytoplasmic structures of gonadotropic cells was demonstrated. The level of detection of intracellular antigens with this method is not as high as that detectable with light microscopical examination of the same specimens, that is, more reaction product at the site of antigen is required to be detectable with scanning electron microscopy than with light microscopy. In spite of the lack of high sensitivity, the intracellular antigens may be localized with the method described.