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

Subconfluent layers of SV24 cells were grown on glass coverslips or multiwell glass slides (Dunn Labortechnik GmbH, Asbach, Germany), rinsed in PBS and fixed with 3% (wt/vol) paraformaldehyde in PBS for 15 rain. After blocking the unreaeted aldehyde groups with 50 mM NI-hCI in PBS, the cells were permeabilized with 0.1% (wt/vol) Triton X-100 in PBS for 5 rain. To prevent unspecific binding of antibodies, the cells were incubated for 30 min with 10% (vol/vol) goat serum in PBS. Primary antibodies were diluted in 10% (vol/vol) goat serum in PBS, and incubated with the cells for 20 rain. After extensive washing with PBS the cells were incubated again for 30 rain with 10% (vol/vol) goat serum in PBS followed by fluorescent labeled secondary antibodies diluted in 10% (vol/vol) goat serum in PBS. The secondary antibodies were used at the manufacturer's recommended dilution. The cell nuclei were stained with Hoechst 33258 stain at dilution 1/1,000 in PBS, to aid in finding the cells in the fluorescence microscope. Cycloheximide chase was done by incubating the cells on coverslips with 20 #g/ml cycloheximide in normal growth medium before the fixation. To see the effect of BfA on the distribution of HBsAg within the cells, the subconfluent cell layers on coverslips were incubated in normal medium supplemented with 10 #g/ml BfA for 2 h, and the cells were fixed and labeled for immunoflunrescence as described above. The immunolabeled ceils were examined with Zeiss Photomicroscope IU with a Planapo 63• oil immersion objective and Zeiss Axiophot microscope with 63x Planapo oil immersion objective. Photographs were recorded on Kodak TMAX400 black and white negative film.