Mouse anti human mmp 9

The supernatant from whole-cell lysates was harvested and immunoprecipitated using the indicated primary antibodies or non-specific IgG as a negative control, which was precleared by protein G magnetic beads at 4°C overnight. Proteins were separated on 10 or 12% SDS-PAGE gels and transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): mouse anti-human Gal-1, rabbit anti-human H-Ras, rabbit anti-human ERK, mouse anti-human p-ERK, rabbit anti-human c-Jun, mouse anti-human MMP-9, rabbit anti-human p21, rabbit anti-human Bcl-2, and rabbit anti-human Raf-1 antibodies. Rabbit anti-human p-AKT (Ser473), rabbit anti-human p-Raf-1 (Ser338), and rabbit anti-human MMP-2 antibodies were purchased from Cell Signaling (Danvers, MA, USA). The mouse anti-GAPDH antibody was purchased from KangChen Bio-tech (Shanghai, China). All experiments were repeated at least twice.

Monoclonal rat anti–mouse CD11b (eBioscience), mouse anti–human CD74 (Abcam), mouse anti–human macrophage surface antigen (AM-3K/CD163), mouse anti–human mannose receptor (CD206; Abcam), and mouse anti–human MMP9 (Santa Cruz Biotechnology, Inc.) monoclonal primary antibodies were used to perform immunofluorescence assays. The Mouse on Mouse kit (Vector Laboratories) was used to reduce background staining when using mouse monoclonal antibodies on mouse tissue sections (n = 3 muscles per group). Goat anti–mouse (Invitrogen) and goat anti–rat (Invitrogen) secondary antibodies conjugated with Cy3 or Alexa Fluor 488 were used. Nuclei were stained with DAPI (OCHEM). The images were acquired using a fluorescence microscope (BX61; Olympus).