Polyclonal rabbit anti mouse ig hrp p0260

Lung tissue was lysed (RIPA buffer; 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) and protein concentration was measured with bicinchoninic assay (BCA, Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of total protein (20 µg) were separated by SDS-PAGE on a Bio-Rad Criterion cell using Criterion precast gels (Bio-Rad, Hercules, CA, USA). Proteins were electroblotted onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich) and incubated with an antibody against eNOS (1:300, ABCAm, Cambridge, UK) in 1% BSA overnight at 4℃. After incubation with the secondary, peroxidase-coupled antibody (Polyclonal Rabbit Anti-Mouse Ig/HRP P0260; DakoCytomation, Carpinteria, CA, USA) detection was accomplished by using enhanced chemiluminescence solution (Western Lightning reagent; Perkin Elmer, Boston, MA, USA) and a ChemiDoc XRS chemiluminescence detection system (Bio-Rad). Densitometric eNOS quantification was accomplished using NIH ImageJ software (1.47, Bethesda, MD, USA). Results were normalized to the respective GAPDH bands.