Goat anti rabbit horse radish peroxidase hrp conjugated secondary antibody

Pericytes were harvested and lysed in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) and centrifuged at 15,000 rpm for 15 min at 4°C. The membranes were blocked with 5% nonfat milk for 1 h and then incubated overnight at 4°C with primary antibody: rabbit anti-Bcl-2, (#2870; 1 : 1000, CST); rabbit anti-Bax, (#2772; 1 : 1000; CST); rabbit anti-Apelin (ab125213; 1 : 500; abcam); and rabbit anti-APJ (ab84296; 1 : 1000, abcam). The membranes were incubated with goat anti-rabbit horse-radish peroxidase- (HRP-) conjugated secondary antibody (1 : 3000, DAKO, Japan) for 1 h at room temperature. The density of each band was analyzed with Image J software. Each experiment was repeated at least three times.

Rat lung Paraffin sections (5 μm thick) were obtained following in vivo experiments as previously described [19 (link)]. Sections were incubated with peroxidase blocking solution (Dako, Cambridge) and then with primary antibodies for rabbit anti-active caspase-3 (1:50 Abcam ab2302), NF-κB p65 (1:200 Cell Signaling C22B4), rabbit anti-phospho-IKKα/β (1:40 Cell Signaling 2697), P-Stat3 (1:50 Cell Signalling 9145); Stat3 (1:400 Cell Signalling 9149) or SMA (1:400 Dako M0851). Sections were then incubated with polyclonal goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) followed by incubation with diaminobenzidine (DAB) and peroxide buffer (Sigma) to produce a brown stain. Slides were counterstained with hematoxylin or eosin to provide nuclear and morphological detail. Non- specific rabbit IgG (Sigma-Aldrich) at the same concentration as those used above was used as a control.