Immunofluorescence Microscopy of HCV

S10-3 cells were seeded onto glass coverslips and transfected 24 h later with ORF3 expression vectors or co-transfected with pUHD15-1 [51 (link)] and pUHD-Cp7 plasmids allowing the expression of HCV core-p7 region, as described previously [30 (link)]. Forty-eight h post-transfection cells were fixed with 2% paraformaldehyde (Sigma-Aldrich) for 10 min and then permeabilized, totally, with either 0.5% saponin or 0.2% digitonin (Sigma-Aldrich) or selectively with 0.01% digitonin. Cells were then washed and incubated 15 min at 20°C in blocking buffer containing 3% bovine serum albumin in PBS. Indirect immunofluorescence was then performed by one-hour incubation at 20°C with primary antibody, followed by 3 PBS washes and incubation with the secondary antibody as described above.