A method for staining intracellular antigens in thin sections with ferritin-labeled antibody.

Abstract

Localization of macromolecules in biological specimens by the use of ferritin-conjugated antibody was first investigated by Singer and Schick (1961), and since that time the technique has been used to stain extraceUular antigens by several workers (Baker and Loosll, 1966; Easton et al., 1962; Morgan et al., 1961). The identification of intracellular antigens was hampered by the lack of adequate penetration of the conjugated ferritin into cells. It was therefore necessary to disrupt the cell membranes by various methods such as dissection of tissue with a razor blade (Andres et al., 1962) or by freezing and thawing (Morgan et al., 1963) to allow access of the ferritinconjugated antibody to the intraceUular antigens. A better method would be the application of the ferritin-conjugated antibody to thin sections of embedded cells so that those antigens on the surface of the sections would be able to combine with the antibody. Two main problems in applying this technique are the destruction of the antigenic determinants by the fixation and embedding and the nonspecific attachment of conjugated antibody to the surface of the embedding plastic (Striker et al., 1966). These problems have been partly overcome by McLean and Singer (1964) by the use of cross-linked polyampholyte and, more recently, by the use of cross-linked bovine serum albumin (McLean and Singer, 1970). A review of studies on the problems of staining of thin sections with the immunoferritin technique has been presented by Sternberger (1967). We report here the results of experiments dealing with the staining of intracellular antigens in thin sections of cells fixed in formaldehyde and erabedded in the water-soluble medium, glycol methacrylate (GMA). 1 In the one case the cells were adenovirus-infected cells (Shahrabadi and Yamamoto, 1970), and in the other the cells were either Micrococats sodonensis or purified cell walls of M. sodonensis.