Van gieson staining solution

Penis tissues were fixed by perfusion with 4% paraformaldehyde and then embedded in paraffin. The specimens were sliced at a thickness of 5 μm, deparaffinized, rehydrated, and subjected to histochemical analysis. Elastica van Gieson staining was performed according to the standard method. In brief, the specimens were stained with resorcin fuchsin staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan), hematoxylin staining solution, and van Gieson staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan). For immunohistochemistry, sections were incubated with a primary antibody reactive to VE-Cad (1:400; LS-B2138; LifeSpan BioSciences), SMA (1:200; ab18147; Abcam) or nNOS (1:400; ab76067; Abcam). Sections were then incubated with biotinylated secondary antibody prior to horseradish peroxidase-labeled streptavidin according to the manufacturer's instructions (DAKO, Cambridgeshire, UK).

The penis was fixed using perfusion with 4% paraformaldehyde. It was then embedded in paraffin and sliced into 5-μm cross sections that were deparaffinized, rehydrated, and subjected to the Elastica van Gieson stain. Elastica van Gieson stain was performed by a standard method. In brief, the specimens were consecutively stained with resorcin‐fuchsin staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan), hematoxylin staining solution, and van Gieson staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan). For immunohistochemistry, sections were incubated with a primary antibody reactive to neural nitric oxide synthase (nNOS: 1:400, Abcam, ab76067) and heterochromatin protein-1γ (HP-1γ: 1:400, Abcam, ab66617). Sections were subsequently incubated with biotinylated secondary antibody and finally horseradish peroxidase-labeled streptavidin according to the instructions provided by the manufacturer (DAKO, Cambridgeshire, UK).