Rat anti mouse f4 80 antibody

Rat anti-mouse F4/80 antibody was purchased from Serotec; Rat anti-mouse CD31 was purchased from Dianova (Hamburg, Germany). Anti-actin and anti-smooth muscle actin (SMA) monoclonal antibodies were purchased from Sigma (St. Louis, MO). Antibodies directed against phopsho-AMPK alpha, phospho-p38 and phospho-JNK, histone H3 and H3K4-di (H3K4me2) and -tri (H3K4Me3) methylation, H3K27 tri-methylation and acetylated H2BK5 were purchased from Cell Signaling Technology (Beverly, MA). Anti-WDR5, anti-BiP, and anti-Ly6g antibodies were purchased from Abcam (Cambridge, UK). The selective inhibitors of LSD1 (GSK-2879552), WDR5 (OICR-9429), MKL1 (CCG-203971), CXCR2 (SB 225002), p38 (SB208530), and JNK (sp600125) were purchased from ApexBio (Boston, MA) and were dissolved in DMSO as stock solutions. DMSO (Sigma D2438) was added to the cell culture medium as control. The heparanase inhibitors H1001 and PG545 were kindly provided by HepaRx Ltd (Ness-Ziona, Israel) and Zucero Therapeutics (Darra, Queensland, Australia), respectively. Mouse CXCL2/MIP-2 Quantikine ELISA kit was purchased from R&D Systems (Minneapolis, MN). The MyD88 peptide inhibitory set was purchased from Novus Biologicals (Centennial, CO). Latent heparanase was purified from medium conditioned by CHO cells overexpressing heparanase essentially as described (25 ) and was added to cell cultures at 1 μg/ml.

Non-parenchymal cells from mouse liver were isolated by 2-step collagenase perfusion (24 (link)). Macrophage positive selection from non-parenchymal cells was performed using QuadroMACS column separation Kit (Miltenyi Biotech, Cambridge, MA). Anti-mouse F4/80 antibody (Biolegend, San Diego, CA) and specific microbeads were used according to the manufacture’s instruction. After the column separation of macrophages, cytospin was performed. Cells were centrifuged at 500 rpm for 5 minutes on glass slides followed by fixation with 4% paraformaldehyde for 10 minutes. Rat Anti-mouse F4/80 antibody (AbD Serotec, Raleigh, NC) was used for immunofluorescent staining as described elsewhere (25 (link)).
Additional methods in online supplement

Inoculated skin was harvested from three to four mice per group at each time point. To detect the presence of rVACV-GFP, skin was preserved in formalin, embedded in paraffin, sectioned, and stained with anti-GFP antibody (Clone JL-8; Clontech, Palo Alto, CA) as previously described (8 ). To detect F4/80 or late viral antigen B5R expression, skin samples were fixed in formalin, embedded in paraffin, sectioned (10 μm) and treated with 1× Target Retrieval Solution (Dako Cytomation). Sections were incubated with purified rat anti-mouse F4/80 antibody (AbD Serotec, Raleigh, NC, USA) or rat MAb19C2, (kind gifts from Bernard Moss, National Institutes of Health), followed by anti-rat horseradish peroxidase-conjugated antibody (Envision detection kit, DAKO). Images were obtained with a Nikon E600 microscope and a Nikon EDX-35 digital camera.