|
|
Fixative |
10% formalin |
Embedding |
Paraffin |
Negative Control |
No primary antibody |
Pretreatment |
Target retrieval 25 min (Steam) |
Blocking |
3% H2O2, 2% Normal Goat Serum |
Primary Antibody |
Anti-Bradykinin Serum (Catalog No.: H-009-01) |
Optimal Dilution |
1:500, 1 hour at RT |
Secondary Antibody |
Goat Anti-Rabbit IgG, Biotinylated (1:400), 30 min |
Amplification |
Streptavidin-HRP (Vector), 1:400, 30 min |
Detection System |
HRP |
Substrate |
DAB (Sigma), 3 min |
Counterstained |
Hematoxylin, 30 sec |
Role of BK in nitric oxide (NO) formation on BPAEC. BPAEC were preincubated with lisinopril (0.5mM) follow by incubation with 100nM HK and PK in HEPES-Tyrode gelatin buffer containing 2mM Ca2+, 1mM Mg2+, 8µM Zn2+, and 0.5mM lisinopril at 37C for 30min in the absence or presence of 100µM antipain, 5µM B2 BK receptor antagonist (B2 INHIBITOR, HOE 140, D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg, Catalog No. 009-18, from Phoenix Pharmaceuticals), 5µM B1/B2 BK receptor antagonist (B1/B2 INHIBITOR, D-Arg-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-Arg, Catalog No. 009-22, from Phoenix Pharmaceuticals), 0.2 to 0.5mM NG-nitro-L-arginine methyl ester (L-NAME), or 10 to 20µM NG-monomethyl-L-arginine (L-NMMA). At the end of the incubation, the wells were washed and the amount of NO formation was measured as indicated in the METHODS. Data are means ± SE of five determinations.
Zhao et al. Am J Physiol Heart Circ Physiol 2001 Apr;280(4):H1821-9 |
