Nanozoomer digital pathology view2

To assess cell proliferation within tumor nodules, liver sections were immunostained for Ki67, and immunofluorescence (IF) images were captured by NanoZoomer and analyzed by NanoZoomer Digital Pathology (NDP) view2 software (Hamamatsu Photonics, Shizuoka, Japan). Software sources and versions are listed in Supplementary Table S3, and antibodies used for IF are in Supplementary Table S4. The number of Ki67+ nuclei were counted in 8–10 random fields for each specimen. Lipid peroxidation in DEN-treated liver tissues was evaluated by IF staining of 4-hydroxynonenol (4-HNE). Images were captured using the Axioskop 2 microscope (Carl Zeiss Canada Ltd., Toronto, Canada), and mean fluorescence intensity (MFI) was quantified (8–10 random fields/section; 3–5 mice per genotype) using the Image J software (National Institutes of Health, Bethesda, MD, USA).

Mouse tissues were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (3 μm). Immunohistochemistry (IHC) was performed using a VENTANA Discovery Ultra automated staining instrument (Ventana Medical Systems, Tuscon, AZ, USA) and VENTANA reagents, according to the manufacturer’s instructions. Sections were then incubated with antibodies specific for SOX9 (Ab185966, Abcam) and were counterstained with haematoxylin for 4 min and dehydrated before coverslip addition. Histology and IHC slides were scanned with a Nanozoomer Hamamatsu device (Hamamatsu Photonics, Tokyo, Japan) and analyzed with Nanozoomer Digital Pathology (NDPview2) software (Hamamatsu Photonics).