NOS Enzyme Levels

Cavernosal tissues were excised, and the urethral tissues were excluded. The cavernosal tissues were diced into small pieces in cold PBS and homogenized using a homogenizer (Ultra Turrax Type T25-B, IKE Labortechnic, Germany) at 16,000 rpm for 3 min on ice in buffer. Afterwards, they were rehomogenized with a glass homogenizer on ice, and then they were subjected to two freeze thaw cycles followed by ultrasonication. The resulting homogenates were centrifuged at 5000 rpm for 30 min at −20°C. Protein levels of NOS isoforms (nNOS, eNOS, and iNOS) were evaluated using commercial ELISA kits (Cloud-Clone Corp., Houston, TX) at 450 nm using a spectrophotometer (BioTek ELISA reader model, Winooski, VT) by using 100 μL homogenate. Results were calculated in ng/ml.