The organization of endoplasmic reticulum export complexes

NRK cells grown on 35-mm tissue-culture dishes were infected with vesicular stomatitis virus (strain ts045), postinfected for 4 h at 39.5°C, and permeabilized as described (Plutner et al.. 1992) . After incubation at the permissive temperature (32°C) as described in the Results, the cells were fixed for 30 min with 3% paraformaldehyde and 0.1% GA in PBS (pH 7.4), washed for l0 rain in PBS containing 0.05 M glycine, scraped, mixed with a preheated (40°C) 10% gelatin in PBS, and centrifuged at 15,000 g in a microcentrifuge for 10 min. Cells embedded in gelatin were cooled on ice, and a solid pellet was cut into I-ram-wide cubes. After overnight cryoprotection by infiltration with a mixture of 2.3 M sucrose in 0.1 M phosphate buffer (pH 7.4) containing 20% polyvinyl pyrrolidene, the cubes were mounted on aluminum nails and frozen in liquid nitrogen. Ultrathin cryosections cut on a Reichert Ultracut E, equipped with a FC-4 cryoattachment, were picked up with 2.0 M sucrose-1% BSA in PBS and collected on Formvar/carbon-coated nickel grids. Sections were then quenched in 0.01 M glycine in PBS, incubated for 30 min in 10% FBS-PBS at rt, and for 1-2 h with primary antibodies diluted in 10% FBS-PBS antibody. Excess primary antibody was removed by multiple rinses in 5% FBS-PBS, followed by transfer of the section to a drop of 10% FBS-PBS containing 6 or 10 nm gold--conjugated anti-rabbit antibodies. After a 2-h incubation at rt, the grids were washed in double-distilled water and stained in 2% neutral uranyl acetate (10 rain), followed by embedment in 3.2% polyvinyl alcohol/0.2% methyl cellulose~ containing 0.2% uranyl acetate. No labeling was observed in controls in which primary antibodies were omitted.