Ab80508

The cryopreserved seminal vesicles were ground and incubated with lysis buffer to extract the protein. 40 µg protein lysate of each sample was electrophoresed in sodium dodecyl sulfate/polyacrylamide (SDS-PAGE) gel and transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). Then the membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich; V900933) for 1 h and incubated with the primary antibodies at 4 °C overnight, including antibodies of Bax (1:500; Affinity, AF0120, Wuhan, China), Bcl-2 (1:500; Affinity, AF6139), NOX1 (1:500; Abcam, ab55831), NOX2 (1:500; Abcam, ab80508), NOX4 (1:500; Servicebio, GB11347, Wuhan, China),and β-actin (1:500; BM0627; Boster, Wuhan, China). After incubation with horseradish peroxidase-conjugated secondary antibodies (1:1000; 7074, 7076; CST, Danvers, MA, USA), the membranes were developed using Bio-Rad Clarity Western ECL Substrate (1705061; Bio-Rad Laboratories, Hercules, CA, USA). Protein bands were analyzed with Image J software (National Institutes of Health, Bethesda, MD, USA).

Immunohistochemistry was performed to detect the content and location of NOX1, NOX2 and NOX4 in seminal vesicle. The paraffin-embedded tissues were cut into sections. After dewaxing and dehydration, the sections were incubated with 3% hydrogen peroxide to inactivate the endogenous peroxidase, followed by antigen recovery with boiled citrate buffer. The sections were then washed with phosphate buffer saline and blocked with goat serum. Sections were incubated with antibodies against NOX1 (1:200; Abcam, ab55831), NOX2 (1:200; Abcam, ab80508), and NOX4 (1:200; Servicebio, GB11347) at 4 °C overnight. After that, sections were incubated successively with biotinylated secondary antibody, peroxidase-conjugated streptavidin, diaminobenzidine and Harris’s hematoxylin. Images were observed with a microscope (Olympus).