Ventana automated staining platform

For immunolocalization of cleaved caspase-3 in liver tissues, paraffin-embedded mouse liver sections (5 µm) were dried 1 h at 58 °C, followed by antigen retrieval and incubated with primary antibody (Cell Signaling, 9661S) in a Ventana automated staining platform (Ventana Medical Systems, USA). Revelation of primary antibody was carried out using horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, USA) and DAB substrate kit (Ventana, #760-124). Slides were then counterstained with hematoxylin. TUNEL analysis was performed on paraffin-embedded mouse liver sections (5 µm), incubated after antigen retrieval with a mix, composed of terminal transferase (Roche, #3333566011) and digoxigenin-11-UTP (Roche, #1558706) followed by HRP-anti-digoxigenin (Ventana, #760-4822). Revelation was used according to the manufacturer’s instructions with the Discovery Rhodamine kit (Ventana #760-233) followed by nucleus labelling with DAPI.
All paraffin-embedded mouse liver sections were scanned with a digital slide scanner (Hamamatsu, Nanozoomer 2.0-RS) and files were analysed with the NDP viewer software. Quantification of cleaved caspase-3 positive-signal was performed with an image analysis software (NIS-Element AR analysis software, Nikon, Tokyo, Japan) and measured to cover an area of 3.9–5.7 mm².

Mouse liver samples were fixed in 4% paraformaldehyde and embedded in paraffin for immunohistochemistry. For histopathology, hematoxylin and eosin (H&E) staining of liver tissues was carried out to investigate the liver injury. For immunolocalization, paraffin-embedded mouse liver sections (5 µm) were dried for 1 h at 58 °C, followed by antigen retrieval and incubation with primary antibody (anti-cleaved caspase-3, Cell Signaling Technology, #9661, Danvers, MA, USA, or anti-CD45 antibody, BioLegend, #103107, Amsterdam, Netherlands) in a Ventana automated staining platform (Ventana Medical Systems, Illkirch-Graffenstaden, France). Revelation of primary antibody was carried out using horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, Agilent Technologies, Les Ulis, France) and DAB substrate kit (Ventana Medical Systems, #760-124, Illkirch-Graffenstaden, France). Slides were then counterstained with hematoxylin. All paraffin-embedded liver sections were scanned with a digital slide scanner (Nanozoomer 2.0-RS, Hamamatsu Photonics, Massy, France) and files were analyzed with the NDP viewer 2.5 software (Hamamatsu, Hamamatsu City, Japan). Signal quantifications were performed with an image analysis software (NIS-Element AR analysis software, Nikon, Tokyo, Japan).

Mouse liver was collected after slaughtering. Liver fragments were fixed in 4% paraformaldehyde and embedded in paraffin. Sections of 4 µm were used for hematoxylin-and-eosin (H&E), Sirius Red and immunohistochemistry stainings. For immunolocalization of glutamine synthetase (GS, Abcam, ab73593, 1/100), or of CD45 (BioLegend, #103107, 1/30), tissue sections were dried 1 h at 58 °C, followed by antigen retrieval and incubated with the corresponding primary antibody in a Ventana automated staining platform (Ventana Medical Systems, Illkirch-Graffenstaden, France). Revelation of primary antibody was carried out using horseradish peroxidase (HRP)-conjugated secondary antibody (Dako, Agilent Technologies, Les Ulis, France) and DAB substrate kit (Ventana, #760-124). Slides were then counterstained with hematoxylin.
All paraffin-embedded liver sections were scanned with a digital slide scanner (Nanozoomer 2.0-RS, Hamamatsu Photonics, Massy, France) and files were analyzed with the NDP viewer 2.5 software (Hamamatsu). Quantification of Sirius Red, CD45 or glutamine synthetase positive signals was performed with an image analysis software (NIS-Element AR analysis software, Nikon, Tokyo, Japan).