VISUALIZATION OF EPIDERMAL GROWTH-FACTOR RECEPTOR IN CRYOSECTIONS OF CULTURED A431 CELLS BY IMMUNO-GOLD LABELING

Abstract

Cryoultramicrotomy in combination with immuno-Au labeling has been demonstrated to present a powerful tool in the visualization of extra- and intracellular located antigens. This method was used to localize epidermal growth factor (EGF) receptor in cultured A431 human epidermoid carcinoma cells. Both the labeling efficiency, maintenance of antigenicity, and the recognizability of the ultrastructure in cryosections are highly dependent upon the fixation procedures. Using 125I-EGF or a consecutive labeling with a monoclonal anti EGF-receptor antibody, rabbit-anti-mouse antibody and 125I-protein A, it was shown that maintenance of antigenicity was optimal using 2% paraformaldehyde as a fixative; under these conditions also the recognizability of ultrastructure was sufficient. After appropriate fixation and labeling, Au particles were observed associated with various regions of the plasma membrane, including coated pits, and with various types of vesicles, including coated vesicles, intracellular vesicular membranes, multivesicular bodies and lysosomes. The results indicate that this method allows a visualization of EGF-receptors and resolution of the EGF-receptor processing pathway at the EM level, independent of the internalization process of labeled ligands.