Molecular Signaling in Penile Tissue

Animals were sacrificed by intraperitoneal injection with sodium pentobarbital (50 mg/kg), followed by bilateral thoracotomy. Penile midshaft tissues were freshly harvested and fixed [23 (link)], and immunofluorescence staining was performed as previously described [31 (link)]. The corpus cavernosum paraffin sections were immunostained with VEGF (diluted 1 : 200 Santa Cruz Biotechnologies, Santa Cruz, US), neuronal nitric oxide synthase (nNOS, diluted 1 : 200 Santa Cruz Biotechnologies, Santa Cruz, CA), brain-derived neurotrophic factor (BDNF, diluted 1 : 200; Abcam, Cambridge, UK), nerve growth factor (NGF, diluted 1 : 200; Abcam, Cambridge, UK), stromal cell-derived factor-1 (SDF-1 diluted 1 : 200; Abcam, Cambridge, UK), and neuron-specific β-III tubulin (diluted 1 : 200; Abcam, Cambridge, UK) and mounted with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA) to stain the nuclei. The collagen and vascular smooth muscle was evaluated after tissue staining with the Masson trichrome technique using the method previously described [32 (link)]. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2009 (Zeiss).