Tissue samples obtained from the peritoneum of mice were fixed in 4% phosphate-buffered formaldehyde and then paraffin processed and embedded. Cut sections were stained with Masson’s trichrome stain. Tissues from the skin inflammation mouse model were formalin-fixed and paraffin-embedded, then sectioned for H&E staining with image capture by Leica DM IL microscope. Alternatively, skin sections were deparaffinized and antigen retrieval was performed by heating in citrate buffer (pH 6.0). Endogenous peroxidases were inhibited by incubation with 0.9% hydrogen peroxide and blocking was performed using 3% BSA. Sections were incubated with mouse anti-Ly6G antibody (Bio X cell), or Anti-S100A9 (Cell Signalling Technology). Antibody binding was detected with an HRP-labelled secondary antibody (Santa Cruz Biotechnology Inc.) and visualised by the DAB+ Substrate Chromogen System (Dako Omnis). Samples were counterstained with hematoxylin, incubated in ethanol and xylol solutions of ascending concentrations then mounted. Images were captured with a Nikon DS-F2 microscope or stained slides were scanned at 20 x magnification using the Aperio ScanScope XT imaging system (Aperio, Vista) and analysed using the ImageScope software.
