Neuron specific β 3 tubulin

The following primary antibodies were used: neuron-specific β-III tubulin (diluted 1:200, Abcam, Cambridge, UK), neuronal nitric oxide synthase (nNOS, diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), α-smooth muscle actin (α-SMA, diluted 1:500; Abcam), VEGF (diluted 1:200; Santa Cruz Biotechnologies) and 6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA) was used to stain nuclei. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2012 (Zeiss).

To prepare paraffin blocks, fresh penile midshaft tissues were harvested and fixed in 4% paraformaldehyde for 24 hrs at 4 °C. Corpus cavernosum paraffin sections were immunofluorescence stained with neuron-specific β-III tubulin (diluted 1:200; Abcam, Cambridge, UK), stromal cell-derived factor-1 (SDF-1 diluted 1:200; Abcam, Cambridge, UK) and neuronal nitric oxide synthase (nNOS, diluted 1:200 Santa Cruz Biotechnologies, Santa Cruz, CA, USA). To stain nuclei, incubating with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA, USA) was performed. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany). Mean intensity was then calculated using ZEN 2009 (Zeiss).