Fluorescein isothiocyanate conjugated goat anti mouse igg

HepG2 cells cultured on coverslips were incubated with p239-b or p239 for 30 min at 4 °C, and then directly harvested or harvested after 1, 8 or 32 h re-culturing at 37 °C. The cells were fixed with 4% paraformaldehyde (Sigma Aldrich) and permeabilized with 0.3% Triton X-100 (Amresco) in PBS. The samples were blocked with 10% goat serum in PBS for 1 h, incubated with 15B2 (anti-p239 mouse mAb) for 30 min, and then labelled with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Molecular Probes) for 30 min, whereas p239-b was directly incubated with streptavidin APC (Molecular Probes). After three washes, the cells were stained with 0.5% 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Fluorescence signals were detected with a laser-scanning confocal microscope (LSM 780, Carl Zeiss, Germany).

Cells were washed once and fixed with 4% paraformaldehyde (Sigma Aldrich, St. Louis, MO, USA) for 30 min and permeabilized with 0.3% Triton X-100 (Amresco) for 15 min in PBS. After blocking in 2% goat serum diluted in 2% BSA for 1 h, cells were incubated with mAb 4# for 1 h and labeled with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Molecular Probes) for 1 h. After adding DAPI (4,6-Diamidino-2-Phenylindole, Dihydrochloride) (Invitrogen, Carlsbad, CA, USA). The stained cells were visualized with Opera Phenix (PerkinElmer, Inc., Waltham, MA, USA) and automatically counted.

IFA was performed as previously described8 (link)9 (link). We seeded 100,000 HepG2/C3A cells per well onto eight-well Lab-Tek II CC2 slides (Nunc) 1 d before infection. Virus was produced from cells that had been transfected with infectious cDNA clones of the HEV strain Kernow (clone P6; GenBank accession no. JQ679013)29 (link). Then, 100 μL of the mixture of virus and serial eight-fold dilutions (beginning with a dilution of 1:2) of sera were added to each chamber and incubated for 5 h at 34.5 °C in a CO₂ incubator. The virus mixture was removed; the cells were washed with PBS; and fresh medium supplemented with 2% DMSO, 100 U penicillin ml⁻¹, 0.1 mg streptomycin ml⁻¹ and 0.1 mg gentamicin ml⁻¹ was added, followed by incubation at 34.5 °C for 5 d. The cells on the eight-well chamber slides were fixed with 4% paraformaldehyde (Sigma Aldrich) and permeabilized with 0.3% Triton X-100 (Amresco) in PBS. The samples were incubated with monoclonal antibody (mAb) 4#, which was donated by Dr Youchun Wang30 (link), and labelled with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Molecular Probes). The stained cells were visualized with a fluorescence microscope and manually counted.