The rubella virus E1 glycoprotein is arrested in a novel post-ER, pre- Golgi compartment

For immunogold labeling, cells were fixed with 3% paraformaldehyda, 0.05% glutaraldehyde in 100 mM cacodylate-HC1 buffer, pH 7.4 (1 h), after which they were scraped from the culture dish, pelleted in a microfuge, and cryoprotected by infiltration with 2.3 M sucrose in 0.1 M phosphate buffer, pH 7.4, containing 20 % polyvinylpyrrolidone, and then mounted on aluminum nails and frozen in liquid N2 according to Tokuyasu (60) . Ultrathin cryosections prepared from the pellets were collected on carbon/formvarcoated nickel grids and incubated for 1 h in mouse monoclonal anti-E1 ascites (diluted 1/300 in 10% FCS/PBS), followed by rabbit anti-mouse IgG (1 h) and goat anti-rabbit lgG gold (5 ran) conjugate (diluted 1/50) for 1 h. Grids were stained in 2 % neutral uranyl acetate (20 rain) and absorption stained with 0.2 % uranyl acetate (10 min) in 0.2 % methyleellulose, and 2 % carbowax.