Ab66127

Relative expression levels of the expected proteins in penile tissues were detected by western blot analysis as previously described.³⁴ In brief, a portion of penile tissues was lysed in RIPA buffer containing phenylmethylsulfonyl fluoride. The protein was then harvested, and the concentrations were quantified by the BCA assay. Next, equal amounts of protein were loaded in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrically transferred onto a polyvinylidene fluoride membrane. Afterward, the membrane was blocked with 5% skim milk and incubated with primary antibodies including α‐SMA (1:1000, BIOSS, bs‐10196R), eNOS (1:1000, Abcam, ab66127), nNOS (1:1000, Servicebio, GB11145), Caspase‐3 (1:1000, Affinity Biosciences, AF6311), BAX (1:1000, Servicebio, GB11690), Bcl‐2(1:1000, Proteintech, 26,593‐1‐AP), and GAPDH (1:1000, Proteintech, 60004‐1‐lg) at 4°C overnight. Finally, secondary antibodies were used for hybridization, and the obtained membrane was imaged under a molecular imager ChemiDoc XRS + system (BIO‐RAD). FIJI/ImageJ software was applied to determine the integrated density.

Freshly dissected penile and MPG tissues were harvested and fixed with 4% paraformaldehyde for further histological staining analysis. Masson trichrome staining was first carried out to determine smooth muscle and collagen expression levels in penile tissues. A 5‐μm slice was prepared and stained following the manufacturer's instructions, and the smooth muscle was stained red while the connective tissue appeared blue. For immunofluorescence staining, the penile sections were incubated with primary antibodies including α‐SMA (1:300, Servicebio, GB111364), eNOS (1:500, Abcam, ab66127), nNOS (1:500, Servicebio, GB11145), Caspase‐3 (1:200, Servicebio, GB11009‐1), and the MPG sections were covered by primary antibodies including Tuj1 (1:2500, Servicebio, GB11139), and GFAP (1:2500, Servicebio, GB11096) at 4°C overnight. After rinsing the slices with PBS, secondary antibodies were adopted for 1‐h immersion. Nuclei were stained with DAPI. Images were observed, and the interesting areas were captured under a confocal laser scanning microscope (Zeiss LSM 710) and a fluorescence microscope (NIKON, Ti2). FIJI/ImageJ software (National Institutes of Health) was used to carry out image analysis.