Immunoelectron Microscopic Localization of Cell Surface Antigens on Rat Hepatocytes Detected with Monoclonal Antibodies ( HAM 2 and HAM 4 )

he localization of surface antigens and the binding activity of two monoclonal antibodies, HAM2and HAM4,which recognize the rat major histocompatibility complex (MHC)antigen class I and the rat hepato-renal antigen respectively, on dissociated (free) hepatocytes was examined by light (LM) and electron microscopy (EM), and by radioimmunoassay (RIA). Fixed hepatocytes, fixed before dissociation, and fresh hepatocytes, dissociated by collagenase, were treated by direct staining with HAM2-or HAM4-immunogoldcomplexes (HAM2-goldand HAM4-gold). Someof the directly stained hepatocytes were further mixed with antimouse IgG-gold complex (IgG-gold) to supplement the direct staining. The polarity of the sinusoidal and contiguous faces and the bile canaliculus, i.e. the in situ morphology, was well preserved in the fixed hepatocytes, while the fresh cells had lost the polarity and were round. On the fixed hepatocytes HAM2-goldparticles were distributed predominantly on the sinusoidal face, while HAM4-gold particles were localized on both the bile canalicular and sinusoidal faces. Nodifferent antigen distribution on the fresh cells was detected with the two antibodies. Supplementation by IgG-gold was noticeable in most cases. The extent of binding activity in both the immunogold and RIA experiments was lower in the fixed cells than in fresh cells. These results suggest that HAM2and HAM4are useful monoclonal antibodies for detecting the localization of the MHCclass I antigen and the hepato-renal antigen on the hepatocytes, respectively. An important feature of the hepatocyte is that it has polarity of structure and function. It has three faces: sinusoidal, contiguous, and bile canalicular. The sinusoidal face has a large microvillar surface area, specialized for the exchange of metabolites with the blood. The bile canalicular face has a small microvillar surface area where bile secretion takes place. The contiguous face is a smooth surface where the cells interact with neighboring cells. The existence of biochemical component which might correlate with the different functions occurring at the three faces was suggested by the isolation of liver plasma-membrane sub fractions which have different densities, and by the respective morphologic features and specific enzymes (4, 5, 24). Some other kinds of cell membranemolecules-insulin receptor (3), the receptor for asialoglyco-protein (1 1), and the secretory component for IgA (17)-have been studied biochemically, and the localization of each molecule on the hepatocyte surface can be examined morphologically to some extent (16, 19). However, the details concerning the localization of these molecules on the three faces of the hepatocyte have not yet been studied. The monoclonal antibody technique developed by Kohler and Milstein (12) has accelerated the characterization of cell membrane molecules, primarily on lymphocytes (13, 23). Then, using the immunoperoxidase or immunogold techniques, details concerning the localization of membrane molecules detected with monoclonal antibodies have begun to be examined by electron microscopy. Such combinations of electron microscopical localization of cell surface molecules by the monoclonal antibody technique would contribute greatly to clarifying the molecular functions of the various cell surfaces. Such studies might be especially useful for cells which have highly polarized cell surfaces with different functions, such as intestinal epithelial cells or hepatocytes. Wehave reported 5 different monoclonal antibodies to rat hepatocyte membrane antigens (HAM1, HAM2, HAM3, HAM4, and HAM5) (7, 8 ,21), and have re-