Pannoramic flash 250 scanner

All specimens were isolated, fixed in 4% paraformaldehyde solution for 24 h in situ, processed for paraffin embedding, and cut into 4 µm transverse sections for routine H&E staining. For IHC analysis, tumor sections were incubated with antibody against mouse Ki-67 (Cat. No. 12202; 1:200 dilution, Cell Signaling Technology) at 4 °C overnight for the later immunostaining by using diaminobenzidine (DAB). For TUNEL analysis, liver sections were incubated with TUNEL labeling (marked with Green fluorescence) mix at 37 °C for 60 min, and then double stained with DAPI (marked with Blue fluorescence, Sigma, USA). The sections were photographed with a Nikon fluorescence microscope (ECLIPSE, Ts2R-FL, Tokyo, Japan). The slides were scanned by a Pannoramic Flash 250 scanner (Perkin Elmer, Waltham, MA, USA) and viewed by the Pannoramic viewer software program (3D Histech, Waltham, MA, USA). Quantitative analysis was performed by NIH ImageJ 1.32j software.

Liver samples from both human and mouse HCC were isolated, fixed in 4% paraformaldehyde solution for 24 h in situ, processed for paraffin embedding, and cut into 4 µm transverse sections. H&E and Sirius red staining were performed on the sections. The slides were scanned using a Pannoramic Flash 250 scanner (Perkin Elmer, Waltham, MA, USA) and viewed using the Pannoramic viewer software program (3D Histech, Waltham, MA, USA). Immunofluorescence was conducted to detect the oxidative DNA damage using mouse anti-8-oxoG (sc130914, Santa Cruz, Dallas, TX, USA). After washing, the slides were probed with secondary antibodies conjugated to Alexa Fluor 594 anti-mouse immunoglobulin G (IgG; Cat. No. 33212ES60, 1:200 dilution, YEASEN, Shanghai, China) at room temperature. The sections were captured with a Nikon fluorescence microscope (ECLIPSE, Ts2R-FL, Tokyo, Japan).