Anti hbsag

Tissues were fixed in formalin, dehydrated, and embedded in paraffin. Deparaffinized sections (4-5 μm) were treated with 3% H₂O₂ to block endogenous peroxidase activity, then immersed in boiling 0.01% citric acid (pH 6.0) in a microwave oven for 15 min to enhance antigen retrieval. After cooling, some sections were rehydrated with PBS and stained with hematoxylin and eosin (H&E), the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reagent (R&D systems), or periodic acid-schiff (PAS) or PAS-diastase (Sigma-Aldrich) for liver glycogen according to the manufacturer's instructions. Other sections were treated with the following antibodies: anti-PML (Santa Cruz, 1:25 dilution), anti-HBsAg (Dako, 1;100 dilution), or antibodies raised against the E3 ligases, seven in absentia homolog-1 and -2 (Cell Signalling, 1:50 dilution). Immunohistochemistry was performed with the ABC Kit (Dako). An isotype antibody was also used as a control. Immunohistochemical analyses of archival paraffin blocks derived from patients was approved by the Institutional Review Board of the Koo Foundation Sun Yat-Sen Cancer Center, Taipei, Taiwan.

Western blotting method was in accordance with the standard protocols. We used mouse monoclonal anti-albumin (1:100), anti-AFP (1:200), anti-CK-18 (1:100), anti-HBsAg (1:100) anti-HBcAg (1:100) and anti-β-actin (1:500, Dako). We used goat antibodies to mouse IgG (1:500) and developed the blots by DAB.