Rabbit monoclonal anti-VE-cadherin(cadh5) IgG
Antigen: peptide KMLAELYGSDPQEELII
dehydrated supernatant from bioreactor
reconstitute with 200µl water and add 160µl glycerol for storage at -20°C
Western Blotting: 1:2000-1:20000, cross-reactive with a 160kDa protein in mouse lung
Immunohistochemistry, formalin/paraformaldehyde fixed, paraffin sections:
Tissues were processed with an automated processor and 5µm sections were mounted on slides and baked at 60C for one hour prior to deparaffinization.
Antigen retrieval (25 min in vegetable steamer, citrate buffer, pH 6.0) was performed followed by blocking with 2% normal goat serum and 1% bovine serum albumin (BSA) in PBS, followed by incubation with the avidin/biotin block for 15 min (Sp2001, Vector Laboratories, Burlingame, CA). Slides were washed four times for 10 minutes between incubations with PBS containing 0.5% Tween-20 (PBS-T). Antibody was diluted in 2% normal goat serum and 1% BSA in PBS and incubated at 4°C overnight. Biotin conjugated anti-rabbit IgG (111-065-144, Jackson Immunologicals, West Grove, PA) was used. Endogenous peroxidase was blocked by incubation with 3% hydrogen peroxide in PBS for 15 min. The ABC Elite kit (Pk6100, Vector Laboratories, Burlingame, CA) was used with diaminobenzidine as substrate. Counterstaining was carried out with Gill1 Hematoxylin (72404, Richard Allen, VWR) for 5-10 seconds prior to dehydration and mounting with Permount (Fisher) under coverslips.