Immuno-EM

CV-1 cells were infected with SV40 for 7h and 24 h, fixed chemically (with 3% paraformaldehyde and 0.2% glutaraldehyde), dehydrated with ethanol, and embedded in London Resin (LR) white (Electron Microscopy Sciences, PA) at 55°C for 48 hours. The thin sections (70 nm in thickness) were cut using an ultramicrotome with a diamond knife. The sections were collected onto bare nickel grids. The sections are then stained immunochemically with primary antibodies raised against antigens exposed on the surface of the sections. The primary antibodies are visualized by staining immunochemically with secondary antibodies raised against the species and isotype of the primary antibodies, conjugated to colloidal gold particles. The immunochemically stained sections are then contrast stained with 0.5% uranyl acetate (Electron Microscopy Sciences, PA) to reveal the ultrastructure of the cells followed by carbon evaporation by Leica EM ACE600 high vacuum coater and are finally viewed by TEM (JEOL JEM 1400 Plus).