Ab859

After deparaffinization, rehydration and reduction of endogenous peroxidase activity, antigen retrieval of tissue microarrays was performed via heating in a microwave oven at 100°C for 5 min and in a slide container at 50–60°C for 15 min with 1 mM EDTA (pH 8.0). Tissue microarrays were blocked in 3% BSA at room temperature for at least 30 min. Tissue microarrays were incubated with an Ms mAb to HBV surface antigen (prediluted, ab859; Abcam) in a humidified chamber at 4°C overnight. After removing the excess primary antibody, tissue microarrays were incubated with freshly prepared HRP rabbit/mouse reagent (DaKo REATM EnVisionTM Detection System) for 30 min at 37°C. Immunohistochemical staining was not performed with DAB (DaKo REAL EnVision Detection System) until the desired stain intensity was observed. For counterstaining, tissue microarrays were stained in hematoxylin for 15 s. Tissue microarrays were rinsed with tap water for at least 5 min. Finally, tissue microarrays were air-dried and sealed.

We collected the adjacent liver specimens of the 100 eligible HCC patients, which were embedded as paraffin samples. First, we performed sectioning and HE staining to identify normal liver tissues. Then, the liver tissues were sampled in an area approximately 2 cm from the tumor, and tissue microarrays were made. Finally, the following were used to prepare the reagents for IHC: Ms mAb to HBV surface antigen (prediluted, ab859; Abcam, Shanghai, China), ethanol (100%, 95%, 85%, and 70% solutions were diluted in distilled water), 3% hydrogen peroxide (20 ml 30% hydrogen peroxide mixed with 180 ml distilled water), PBS buffer, EDTA pH 8.0 diluted in distilled water to 1 mM, BSA (Sigma-Aldrich, Shanghai, China), the DaKo REAL EnVision Detection System, deionized water, xylene, and hematoxylin.