Sc8399

The imHCs were cultured onto 96-well CellCarrier-96 optic black plates (PerkinElmer, Waltham, MA, USA) and stained with antibodies against hepatocyte markers: Albumin (ALB) (1:100 dilution, ab10241, Abcam), α-fetoprotein (AFP) (1:100 dilution, SC8399, Santa Cruz Biotech, Dallas, TX, USA), LDLR (1:100 dilution, SC373830, Santa Cruz Biotechnology), sodium taurocholate cotransporting polypeptide (NTCP) (1:100 dilution, ab131084, Abcam), MRP2 (1:100, AB3373, Abcam) and hepatocyte nuclear factor-4α (HNF-4α) (1:100 dilution, SC6556, Santa Cruz Biotech). For detecting HBV infectivity, infected hepatocytes were stained with antibodies against HBV proteins: HBcAg (1:100 dilution, ab8637, Abcam), HBsAg (1:100 dilution, ab20758, Abcam). Hepatocytes were then incubated with goat anti-mouse Alexa Fluor^® 488-conjugated (1:500 dilution, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), goat anti-rabbit Alexa Fluor^® 488-conjugated (1:500 dilution, Invitrogen), or donkey anti-goat Cy3-conjugated secondary antibody (1:500 dilution, BioLegend, San Diego, CA, USA). Hepatocyte nuclei were stained with 2 µM Hoechst 33342 (Thermo Fisher Scientific, MA). Mouse IgG2a, mouse IgG1, rabbit IgG and goat IgG were used as negative control for staining. Fluorescence images were captured by an Operetta High-Content Imaging System (PerkinElmer, MA) with a 40× objective lens.