Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment

For confocal microscopy, subconftuent SV24 cell layers on coverslips were fixed with 3 % paraformaidehyde in PBS as for normal immunofluorescence. Cells were permeabilized with 0.2% Triton X-100 in PBS for 5 rnin. Free aldehyde groups were quenched with 75 mM NI-I4CI and 20 mM glycine in PBS. To prevent unspecific binding of the antibodies the cells were incubated with 0.2% fish skin gelatin (wt/vol) in PBS for 10 min. The pri-mary antibodies were diluted in PBS containing 0.2% fish skin gelatin and incubated with the cells for 30 rain. After washing with PBS the cells were incubated with 0.2% fish skin gelatin in PBS for 10 mill, followed by fluorescent labeled secondary antibodies diluted in PBS containing 0.2% fish skin gelatin. After washing with PBS, the cells were postlixed for 30 rain with 4% paraformaldehyde in cacodylate buffer (pH 7.4). Postfixation was necessary because 1,4-diazabicyclo[2.2.2]octane could cause marked changes in call morphology and redistribute the antibodies unspecifically. The coverslips were mounted on glass slides with 50% glycerol in PBS, containing 100 mg/mi 1,4-diazabicyclo[2.2.2]octane to prevent fluorescein quenching during microscopy. The mounted cells were examined with laser scanning confocal microscope (EMBL Light Microscopy Group) using a single excitation wavelength (514 nm) and selective filters for fluorescein and rhodamine.