Dab staining

Immunohistochemistry was performed on the tissue microarray slide conventionally. In brief, the slide was deparaffinized by xylene and rehydrated using a graded ethanol series. Then, 3% H₂O₂ was used to block endogenous peroxidase in the tissues. Antigen retrieval was completed using microwave treatment. Then, 5% bovine serum albumin was used to block nonspecific reactions.The slides were incubated with primary antibody against E-cadherin (SCBT, Santa Cruz, CA, USA, 1:100), vimentin (SCBT, 1:100) and HBx (US Biological, Swampscott, MA, USA, 1:100) at 4 °C overnight. The streptavidin-biotin peroxidase kit (ZSGB Bio, Beijing, China) was used to detect the bound antibodies. Finally, the band was visualized by DAB staining (ZSGB Bio).
The immunohistochemical result was scored using the intensity and extent. Staining intensity was quantified as follows: negative (0), weak (1), moderate (2) or strong (3). Staining extent was quantified according to the percentage of positive cells: none (0), <25% (1), 25–50% (2), 50–75% (3) or >75% (4). The final score was calculated as the intensity score multiplied by the extent score.

After the rats were sacrificed, fresh myocardial tissue samples were fixed in formalin to prepare paraffin-embedded blocks, which were cut into 5-μm paraffin sections for histological analysis. The paraffin sections were stained with hematoxylin and eosin (H&E), Masson’s trichrome, Sirius red, and wheat germ agglutinin (WGA) to assess the standard histology, distribution of collagen, extent of fibrosis, and morphology of the cardiomyocyte membrane, respectively. For IHC, the paraffin sections were sequentially subjected to dewaxing, dehydration with gradient alcohol, microwave thermal repair in citrate buffer (0.01 M, pH 6.0), endogenous peroxidase blocking, and blocking in 10% goat serum in phosphate-buffered saline. The sections were then incubated with primary antibodies, anti-collagen I (Affinity, 1:1,000) or collagen III (Affinity, 1:500), overnight at 4°C. The next day, sections were incubated with HRP-conjugated anti-rabbit IgG (ZSGB-BIO, Beijing, China), and reactions were detected by DAB staining (ZSGB-BIO, Beijing, China). The nuclei were stained with hematoxylin and differentiated in 1% acid alcohol, after which the sections were rinsed with running water. Finally, the slides were sealed with a neutral gel. Images were acquired using an Axio Scope A1 microscope (Zeiss, Oberkochen, Germany).