Anti p22phox antibody

DPI and 3-bromopyruvate were purchased from Sigma-Aldrich (St. Louis, MO). CM-H2DCF-DA, DAF-FM, HEt, and 2-NBDG were purchased from Invitrogen/Molecular Probes (Carlsbad, CA). SP600125 was acquired from EMD Biosciences (Calbiochem, San Diego, CA). DPI was dissolved in dimethyl sulfoxide (DMSO) and freshly diluted in culture media before used. The final DMSO concentration was less than 0.1% (v/v). In addition, 3-bromopyruvate was dissolved in water and neutralized with NaOH immediately before use in cell culture. The rabbit polyclonal anti-p22^phox antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-Akt and anti-phospho-Akt (Ser473) antibodies as well as rabbit monoclonal anti-c-Jun and anti-phospho-c-Jun (Ser63) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Thoracic aorta sections (5 μm) were sliced from paraffin blocks fixed with 4% paraformaldehyde. Antigen retrieval was performed by heating the sections in a 60 °C oven with a 10 mM citrate buffer overnight. After cooling, the sections were incubated with 3% hydrogen peroxide for 10 min to inhibit endogenous peroxidases. A rabbit polyclonal anti-p22phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at a 1:50 dilution for p22phox immunostaining. An Alexa Fluor 488-conjugated secondary antibody (Invitrogen Corp., Carlsbad, CA, USA) was used at a 1:2000 dilution. For p47phox immunostaining, the sections were incubated with a rabbit polyclonal anti-p47phox antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted at 1:50 in 0.5 M TBST at 4 °C overnight. An Alexa Fluor 555-conjugated secondary antibody (Invitrogen Corp., Carlsbad, CA, USA) was used at a 1:2000 dilution. Nuclear counterstaining was accomplished with DAPI at a 1:10,000 dilution. The sections were stored in the dark until they were analyzed using a confocal microscope (LSM 510 META, Carl Zeiss, Inc., Overkochen, Germany).