Penile Tissue Immunohistochemical Staining

Penile mid-shaft tissues were harvested, fixed and further processed for immunohistochemical staining as previously described [15 (link)]. Briefly, tissues were fixed in cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, pH 8.0, for 4 hours followed by overnight immersion in buffer containing 30% sucrose. The specimens were then embedded in OCT Compound (American Master Tech Scientific, Lodi, CA, USA) and stored at -70°C until use. Sections were cut at 5 μm, mounted into charged slides and air dried for 5 minutes. Representative slides were stained with Masson’s trichrome for connective tissue and smooth muscle histology.
For immunohistochemical examination, tissue sections were stained with mouse anti-rat endothelial cell antigen-1 (RECA-1, Abcam Inc, Cambridge, MA, USA) and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL, Roche Diagnostics Corporation, Indianapolis, IN, USA) using standard techniques [16 (link)]. To visualize ADSC, EdU staining with immunostaining for α-smooth muscle actin (α-SMA) was done with mouse anti-α-SMA (Sigma-Aldrich, St. Louis, MO, USA) and freshly made Click-IT reaction cocktail (Invitrogen, Carlsbad, CA, USA). Nuclear staining was performed with 4’,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) [26 (link)].