Monoclonal mouse anti mmp9

Tissue homogenate was prepared from 7-week-old C57B/6 mice inoculated with 1 × 10⁶ CFU S. aureus subcutaneously. Control tissue was isolated from the same animal. Protein concentration of homogenates was determined using a Bio-Rad protein assay (Hercules, CA). Two μg of protein was loaded onto a 10% SDS-PAGE gel or a 10% SDS-PAGE gel with 0.01% gelatin. MMP-9 was detected with monoclonal mouse anti-MMP9 (Abcam, Cambridge, United Kingdom). The gelatin gel was incubated for 1 h at room temperature in renaturation buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaCl₂, 1 μM ZnCl₂, and 2.5% Triton X-100) and then overnight at 37°C in development buffer (50 mM Tris-HCl buffer, pH 7.4, containing 5 mM CaCl₂ and 1 μM ZnCl₂). The gel was then stained with Coomassie to visualize zones of clearing.

Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin-EDTA solution and foetal bovine serum (FBS) were purchased from Gibco-BRL (Grand Island, NY, USA). Mouse IL-17A and isotype-control Abs were purchased from R&D Systems (Minneapolis, MN, USA). Monoclonal mouse anti-MMP-2, monoclonal mouse anti-MMP-9 and monoclonal rabbit anti-occludin were purchased from Abcam (Hong Kong, China). Monoclonal mouse anti-claudin-5 antibody was purchased from Invitrogen (Invitrogen, USA). Recombinant IL-17A protein and Fluoroshield mounting medium with 4,6-diamidino-2-phenylindole (DAPI) were purchased from Abcam (Hong Kong, China). The mouse IL-17A ELISA kit, IL-6 ELISA kit and IL-1β ELISA kit were obtained from R&D Systems (Minneapolis, MN, USA).

Tissues from the skin abscess or surrounding sterile field were processed for immunofluorescence staining using primary antibodies for matrix metallopeptidase 9 (monoclonal mouse anti-MMP9; Abcam) and collagen type I (polyclonal rabbit anti-ColI; Millipore, Burlington, MA). Frozen tissue sections were fixed (100% methanol), blocked in 5% donkey serum, and incubated with primary antibodies (Arg-1, 1:100; iNOS, 1:100; MMP-9, 1:500; ColI, 1:40) diluted in 2% donkey serum overnight at 4°C. Sections were then incubated with donkey anti-mouse Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 648 (1:250; Jackson ImmunoResearch Laboratories, West Grove, PA) secondary antibodies and Hoechst stain (1:100; Molecular Probes, Eugene, OR) for 2 h at room temperature. The samples were washed and dried, and the glass slides were mounted with Slow Fade (Life Technologies, Carlsbad, CA). Confocal imaging was performed using a Zeiss 710 META laser scanning microscope (Carl Zeiss, Oberkochen, Germany).