Cd68 ed 1

Kidney sections from mice were prepared as described A citrate-based antigen retrieval solution was used. Sections were exposed to 3% H₂O₂ in methanol for 5 minutes to quench endogenous peroxidases. Endogenous mouse Ig staining was blocked with M.O.M^TM mouse IgG blocking regent (Vector Laboratories) for 1 h. Sections were incubated with the CD68 (ED-1, mouse monoclonal; Abcam, Cambridge, MA) or MPO (mouse monoclonal, 1:200; Novus) or anti-P50 (sc-114, rabbit polyclonal 1:5000), anti-cleaved Caspase-3 (#9661, rabbit polyclonal 1:500; cell signaling Technology, Danvers, MA), TLR4 (mouse monoclonal, 1:200; Abcam, Cambridge, MA) overnight at 4 °C for overnight. After washing, sections were incubated with an M.O.M^TM biotinylated anti-mouse IgG or anti-rabbit biotinylated secondary antibody at room temperature for 30 min, and then with the avidin–biotin–peroxidase complex (Vector M.O.M.^TM Immunodetection Kit, Vector Laboratories). The reaction products were developed using the 3, 3′-diaminobenzidine substrate from Vector Laboratory, mounted with a glass coverslip, and photographed using a Zeiss Axioplan2 microscope with a Q-imaging MP3.3 RTV camera under 200 or 400 magnification. The area labeled was quantified using Image-Pro Plus software.