Lab tek 2 cc2 slides

HepG2 cells (4×10⁴) were seeded onto eight-well Lab-Tek II CC² slides (Nunc) one day before infection. IFA for detection of HEV-infected cells was performed as described [62 (link)]. To examine the subcellular localization of MAVS and IRF3, cells were fixed with 4% paraformaldehyde for 20 minutes and permeabilized with 0.2% Triton X-100 for 15 minutes. Cells were stained with pre-absorbed ch1313 serum and a rabbit anti-MAVS, mouse anti-PMP70, or rabbit anti-IRF3 for 1 h, and subsequently incubated with Alexa Fluor 488/594-conjugated goat-anti-rabbit IgG, Alexa Fluor 488/594-conjugated goat-anti-mouse IgG, or Alexa Fluor 488-conjugated goat-anti-human IgG (Invitrogen) for 1 h. After adding antifade-4 6-diamidino-2-phenylindole (DAPI) mounting solution (Sigma), slides were viewed with a Zeiss LSM 510 confocal microscope with a 63x (NA1.2) apochromatic water objective. Images were acquired using the ZEN 2009 software.

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.