Mouse anti hbx monoclonal antibody

Slices of tissue samples were deparaffinized in xylene, dehydrated in graded alcohol series, and washed with distilled water before subjecting to antigen retrieval in the boiling citrate buffer. Then, these slices were rinsed with PBS, incubated with 3% H₂O₂ at room temperature (RT) for 15 min, and blocked with goat serum at RT for 15 min. Mouse anti‐HBx monoclonal antibody (1:200; Abcam, Cambridge, MA) or rabbit anti‐14‐3‐3ζ polyclonal antibody (1:200; Abcam) was used to incubate tissue slices at 4°C overnight. Thereafter, these sections were incubated with biotin‐labeled goat anti‐rabbit or anti‐mouse secondary antibody (1:200; Beyotime, Shanghai, China) at 37°C for 30 min, and then with HRP‐labeled streptavidin at 37°C for additional 30 min. The expression signal was magnified by 100 μL DAB, and the cell nuclei were counterstained with hematoxylin.

Cells were seeded on coverslips and allowed to adhere for 12 h. After treatment, cells were fixed with 100 % methanol for 30 min and blocked with 5 % bovine serum albumin (BSA) for 30 min. Coverslips were incubated with mouse anti-HBx monoclonal antibody (1:100, abcam) or rabbit anti-TLR4 polyclonal antibody (1:100, Santa Cruz Biotechnology) in 1 % BSA at 4 °C overnight, followed by with fluorescence labeled secondary antibody (1:100, Earthox, USA) in 1 % BSA for 30 min at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Images were captured using Nikon A1 Confocal Laser Microscope System and adjusted using NIS-Elements Viewer 4.0 (Nikon, Japan).