Anti s100a9

The liver and lung tissues were immediately fixed in 4% paraformaldehyde. They were embedded in paraffin and 3 µm thick sections were cut. For Immunohistochemical staining, EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC Kit (Abcam, Cambridge, UK) was used. The protocol given by the manufacturer was strictly followed. In brief, the sections were deparaffinized and the endogenous peroxidase activity was blocked using the hydrogen peroxide block (given along with the kit). The blocked sections were incubated with the primary antibodies. The primary antibodies used were: anti-S100A8 (dilution 1:100, Abcam, Cambridge, MA, USA), anti-S100A9 (dilution 1:100, Novus Biologicals, Littleton, CO, USA), anti-TNFα (dilution 1:100, ABclonal, Woburn, MA, USA), anti-NFκβp65 (dilution 1:100, Novus Biologicals, Littleton, CO, USA). The secondary antibody-HRP conjugate is given along with the kit. Primary antibody binding was visualized using diaminobenzidine (given along with the kit, Abcam). The sections were then counterstained using Mayer Haematoxylin and mounted.

Tumor mass explants were embedded in O.C.T., and quickly frozen in liquid nitrogen-cooled isopentane for sectioning at a thickness of 10 μm on a cryostat. Then, slices were processed for immunofluorescence analyses. As primary antibodies, we used anti-S100A9 (89726, Novus biologicals, Littleton, CO, USA), anti-Von Willebrand factor (A0082, Dako-Agilent, Glostrup, Denmark) and anti-p(S139)H2AX (20E3, Cell Signaling Technologies) at 1:200 dilution. Appropriate AlexaFluor-conjugated secondary antibodies (Thermo Fisher Scientific) were used. Hoechst 33342 (Thermo Fisher Scientific) and AlexaFluor 488-conjugated phalloidin (Thermo Fisher Scientific) were used to stain nuclei and actin, respectively, according to manufacturers’ instructions.
For lipid peroxidation analysis, cells were incubated with C11-BODIPY 581/591 (Thermo Fisher Scientific) at a final concentration of 10 μM for 30 min at 37 °C and fixed with 4% paraformaldehyde solution. Lipid peroxidation was analyzed through fluorescent microscopy. Images were acquired using a Nikon Eclipse TE-2000 fluorescent microscope equipped with UV source and CoolSNAP Photometrics CCD Camera (Nikon Instruments Inc., Amsterdam, The Netherlands).