Demonstration of antigens at both sides of plasma membranes in one coincident electron microscopic image: a double-immunogold replica study of virus-infected cells.

We present here a procedure for obtaining high-resolution topographical information about the spatial distribution of antigens at both sides of isolated plasma membranes. HeLa cells grown on coverslips and infected with measles virus served as a model system. Virus glycoproteins appearing at the cell surface were demonstrated by tagging them with rabbit anti-measles antibodies and protein A-gold probes. Cells were stabilized with tannic acid, covered with a cationized coverslip, and then split in potassium-containing buffer. Membranes adherent to the cationized coverslip were fixed in formaldehyde-glutaraldehyde and reacted with mouse monoclonal antibodies against various structural proteins of measles virus. Antibody binding sites at the cytoplasmic surface were visualized either by the antibody bridge method, using normal mouse Ig coupled to gold colloid of different sizes, or by the peroxidase-antiperoxidase procedure. After osmication and critical point-drying, the cytoplasmic surfaces were replicated by platinum-carbon evaporation and examined by TEM without prior cleaning from biological material. This new method permits concomitant localization of antigens present at the inner and outer leaflets of the plasma membrane, and provides high-resolution information about the three-dimensional organization of the cytoplasmic surface.