Immunohistochemical detection of endothelial cell adhesion molecules in mouse kidney

The expression and localization of E-selectin, VCAM-1, and ICAM-1 proteins in mouse kidney were determined by immunohistochemistry (IHC). Five-micrometer acetone-fixed kidney cryosections from snap-frozen mouse kidneys were incubated with Peroxidase Block (EnVision + System-HRP (AEC); DAKO, Carpentaria, Calif) for 10 min to block endogenous peroxidase. For specific protein detection, sections were incubated with primary rat anti-mouse antibodies against E-selectin (MES-1; hybridoma supernatant, 1:10, kindly provided by Dr Derek Brown, UCB Celltech, Belgium), VCAM-1 (1:10, clone MIK-1.9; ATCC, Manassas, Va), ICAM-1 (5 μg/mL; Southern Biotech, Birmingham, AL), and CD31 (1:100, cat. no. #550274; BD Pharmingen, San Diego, Calif, for endothelial all detection, data not shown) all diluted in 5% fetal calf serum (FCS; Sigma-Aldrich, St. Louis, Mo) in PBS for 1 h at room temperature (RT). After washing, sections were incubated with rabbit anti-rat IgG antibody (mouse adsorbed; Vector Laboratories, Burlingame, Calif) diluted 1:300 in 5% FCS/2% normal mouse serum (Sanquin, Amsterdam, The Netherlands) in PBS for 45 min at RT. After washing, sections were incubated with anti-rabbit labeled polymer HRP antibody from the EnVision kit for 30 min at RT. To visualize peroxidase activity, 3-amino-9-ethylcarbazole (AEC) from the EnVision kit was used and sections were subsequently counterstained with Mayer's hematoxylin (Merck, Darmstadt, Germany).