Cells were fixed for 2 d at 4°C with 4% paraformaldehyde (Electron Microscopy Sciences) in 0.25 M Hepes, pH 7.4, washed in PBS, scraped, and embedded in 10% gelatin. Small pieces of the gelatin pellets were infiltrated overnight at 4°C with 2.3 M sucrose in PBS, and then frozen in liquid nitrogen. Gold sections (95 nm thick) were cut using a Leica ultracut ultramicrotome with an FCS cryoattachment at −108°C and collected on formvar- and carbon-coated nickel grids using a 1:1 mixture of 2% methyl cellulose (25 centipoises; Sigma-Aldrich) and 2.3 M sucrose in PBS (Liou et al. 1996 ).
After quenching with 0.1 M NH4Cl in PBS for 10 min, the grids were incubated for 20 min with a solution of 1% fish skin gelatin (FSG; Sigma-Aldrich) in PBS. They were then incubated with anti-HA antibodies (1:100 in PBS-FSG), washed four times for 4 min in PBS, incubated for 30 min at room temperature with rabbit anti–mouse IgGs (1:50 in PBS-FSG; Cappel/ICN Biomedicals), washed another four times for 4 min in PBS, and finally incubated for 30 min at room temperature with 5 nm protein A–gold (from the laboratory of J. Slot, University of Utrecht, Utrecht, the Netherlands) in PBS-FSG. After four final washes in PBS, the sections were fixed in 1% glutaraldehyde in PBS for 10 min, washed three times in water and stained for 10 min at room temperature with 2% neutral uranyl acetate (Electron Microscopy Sciences). After three short washes in water, the sections were infiltrated with a mixture of 1.8% methylcellulose and 0.5% uranyl acetate and air-dried.
After quenching with 0.1 M NH4Cl in PBS for 10 min, the grids were incubated for 20 min with a solution of 1% fish skin gelatin (FSG; Sigma-Aldrich) in PBS. They were then incubated with anti-HA antibodies (1:100 in PBS-FSG), washed four times for 4 min in PBS, incubated for 30 min at room temperature with rabbit anti–mouse IgGs (1:50 in PBS-FSG; Cappel/ICN Biomedicals), washed another four times for 4 min in PBS, and finally incubated for 30 min at room temperature with 5 nm protein A–gold (from the laboratory of J. Slot, University of Utrecht, Utrecht, the Netherlands) in PBS-FSG. After four final washes in PBS, the sections were fixed in 1% glutaraldehyde in PBS for 10 min, washed three times in water and stained for 10 min at room temperature with 2% neutral uranyl acetate (Electron Microscopy Sciences). After three short washes in water, the sections were infiltrated with a mixture of 1.8% methylcellulose and 0.5% uranyl acetate and air-dried.
