Pannoramic midi

Tissue samples were fixed in 4% formalin solution and paraffin-embedded. For hematoxylin and eosin (H&E) staining, tissue sections were deparaffinized in xylene, hydrated in graded alcohol solutions, and stained with H&E. The samples were examined under an automatic digital slide scanner (Pannoramic MIDI; 3DHISTECH, Budapest, Hungary) after mounting. For immunohistochemistry, tissue sections were deparaffinized in xylene, hydrated in graded alcohol solutions, and heated (100 °C) for 15 min in Antigen Retrieval Citra Solution (pH 6.0) for antigen retrieval. For single immunostaining, endogenous peroxidase activity was blocked in a 1% hydrogen peroxide solution (Sigma-Aldrich, St. Louis, MO, USA) in phosphate-buffered saline (PBS) with 0.3% Triton X-100 for 30 min at room temperature. The sections were incubated with the indicated antibodies overnight at 4 °C, and then incubated with the corresponding horseradish peroxidase-conjugated secondary antibody. Finally, 3,3′-diaminobenzidine (DAB; Dako, Agilent, Santa Clara, CA, USA) was used to detect these labeled antibodies and the nucleus was stained with hematoxylin. After rinsing with PBS, the samples were mounted and analyzed using an automatic digital slide scanner (Pannoramic MIDI; 3DHISTECH).

Cancer, heart, and kidney tissues were extracted from each mouse and fixed using 4 v/v% of formaldehyde for one day. The tissues were embedded in paraffin after being dehydrated with a series of gradient ethanol concentrations. The blocked tissues were sliced to a thickness of 3 µm and stained with H&E. The stained slides were observed using slide scanner (Pannoramic MIDI; 3DHISTECH Ltd., Budapest, Hungary) and panoramic viewer (Version 1.15.3; Pannoramic MIDI; 3DHISTECH Ltd., Budapest, Hungary) program.

Fresh human HCC tissues, normal adjacent tissues, and xenografted tumors were fixed using 4% paraformaldehyde for overnight prior to being embedded in paraffin and sectioned at 4 µm thickness using a cryostat. Sections were then dewaxed using xylene and rehydrated prior being incubated with primary antibodies for 1 h, followed by incubation with corresponding secondary antibodies conjugated with horse‐radish peroxidase for 1 h. Visualization was performed using a DAB Kit (DAKO, Beijing, China) under microscope. Nuclei were then counterstained with hematoxylin (Beyotime Biotechnology), followed by dehydration and coverslip mounting. The antibodies used were listed in Table S2 (Supporting Information). Images were taken using Pannoramic Midi (3DHistech, Budapest, Hungary).
For H&E staining, paraffin sections from human HCC tissues and normal adjacent tissues, as well as from mice subcutaneous tumors generated in xenograft experiment (4 µm thickness) were fixed in 10% formalin and washed with 60% propylene glycerol. Samples were then stained with 0.5% hematoxylin‐eosin (Sangon Bio, Shanghai, China) for 3 min followed by dehydration and coverslip mounting. Images were taken using Pannoramic Midi (3DHistech).