Mmp 9 af909

Liver specimens were embedded in Tissue Tec OCT compound (Miles, Elkhart, IN) and snap frozen in liquid nitrogen and immunostaining performed on cryostat sections (0.5 µm) as described previously [19] (link). Appropriate rat primary antibodies against mouse macrophage antigen-1 (Mac-1; M1/70), CD68 (FA-11, Serotec), Ly-6G (1A8) (BD Biosciences, San Diego, CA), MMP-9 (AF909; R&D Systems, Minneapolis, MN) were used at optimal dilutions. Primary antibody was replaced with dilution buffer for negative controls. Bound primary antibodies were detected using biotinylated anti-rat IgG and streptavidin peroxidase–conjugated complexes (Vector Laboratories, Burlingame, CA). Sections from inflammatory tissues known to be positive for each stain were included as positive controls. Sections were evaluated blindly by counting 30 high-power fields per section.

Primary antibodies used for Western blotting (WB), immunofluorescence (IF) staining and flow cytometry (FC) were as follows with dilutions inticated in parenthesis: MPO, clone 8F4 (HyCult) (IF: 1:200); MPO, AF3667, R&D (1:400); MMP9, AF909, R&D (WB 1:1000); the anti-WASH (WB 1:1000) and anti-FAM21 (IF 1:200) antibodies were described before^{25 (link),28 (link)}. The anti-CD63 (clone NVG-2), anti-CD11b (clone M1/70) and anti-Ly6G (clone 1A8, 127610) (flow cytometry) antibodies were from Biolegend (FC 1:50). The anti-neutrophil elastase, Ab68672, was from Abcam (FC 1:50) and phalloidin was from Thermo Fischer. The anti-Rac1 antibody was from Proteintech (24072-1-AP) (WB 1:1000) and the anti-Rac1-GTP from NewEast Biosciences (26903) (IF 1:100); anti-RhoA was from Santa Cruz Biotechnology (SC-418) (WB 1:1000; IF 1:100), anti-Rab21 from Novus Biologicals (NBP1-81544) (IF 1:200) and anti-Arp2 (ab49674) (IF 1:200) was from Abcam. Myc-Tag (9B11) Mouse mAb #2276 (WB:1:1000). Anti-mCherry antibody Abcam, [EPR20579] (ab213511), WB, 1:1000.