Mouse anti hbcag

Immunofluorescence staining was performed as described previously (11 (link)). Paraffin-embedded para-carcinoma tissues from HCC patients were stained with rabbit anti-CD68 (Abcam) and mouse anti-HBcAg (Abcam) Abs. After washing with PBS, tissues were incubated with goat anti-rabbit IgG conjugated to Alexa Fluor-488 and anti-mouse IgG conjugated to Cy3. The slides were viewed using an OLYMPUS inverted microscope.

Human liver organoids were collected and washed three times with cold Ad+++ to remove BME, then fixed with 4% paraformaldehyde for 30 min in ice, and permeabilized using 0.3% (HBcAg, HNF4α, and NTCP) or 1% (albumin) Triton X-100 (Sigma) in phosphate-buffered saline (PBS) for 30 min at RT. For HBsAg staining, cells were fixed and permeabilized in 100% acetone. Specimens were incubated for 2 hr at RT in PBS plus 10% bovine serum albumin (BSA) (Roche) and 0.5% FCS (HBsAg) or PBS plus 0.5% FCS, 0.3% triton, 1% BSA 1% DMSO (albumin, HBcAg, HNF4α, and SLC10A1). Following blocking, human liver organoids were incubated overnight with primary antibodies (mouse anti-HBcAg, mouse anti- HBsAg (ThermoScientific), mouse anti-HBcAg (Abcam)), mouse anti-albumin, rabbit anti-HNF4α (Santa Cruz Biotechnology), and rabbit anti-SLC10A1 (NTCP, Sigma) diluted in PBS + 10% blocking buffer. After extensive washing, human liver organoids were stained with appropriate Alexa Fluor dye-conjugated secondary antibodies (Life Technologies). Nuclei were stained with Hoechst33342 (Molecular Probes). Immunofluorescence images were acquired using a confocal microscope (Leica, SP5). Images were analyzed and processed using Leica LAS AF Lite software (Leica SP5 confocal). All phase-contrast pictures were acquired using a Leica DMIL microscope and a DFC420C camera.