Immunocytochemistry: its evolution and criteria for its application in the study of epon-embedded cells and tissue

Abstract

The word immunocytochemistry is currently used to describe a number of methods that can be employed to localize antigens within cells by means of antigen-specific antibodies. In this article we will review a number of these methods, including immunofluorescence, immunoperoxidase, avidin-biotin, and colloidal-gold techniques. The advantages and disadvantages of the various methods are discussed, special attention being focused upon immunocytochemical staining of plastic-embedded tissue. Studies on the light microscope level show that embedding tissue in plastic prior to immunoperoxidase staining not only improves visualization of antigen-specific staining but also provides an accurate and efficient means of prescreening tissue for antigen prior to immunocytochemical staining on the electron microscope level. Varying section thickness between 1 and 3 microns does not significantly influence staining, whereas the fixative used to preserve the tissue under study does. On the electron microscope level, the colloidal gold technique appears superior to immunoperoxidase staining. It is both esthetically more pleasing and highly sensitive. Of five different colloidal gold methods tested, the most sensitive is the two-step technique that employs an antigen-specific primary antibody followed by a gold-labeled secondary antibody. Throughout this article, special emphasis is placed on the use of proper controls, both on the light and electron microscope levels. Where possible, such controls should include substitution of specific antiserum with normal serum; the use of antigen-adsorbed antiserum; the use of antisera with specificities for antigens not present in the tissue being studied; the use of tissue previously shown to be stainable for the antigen; and if cultured cells are being studied, the use of a number of cell types that do not contain the antigen.