Af5457

After three washes with 0.01 mol/L PBS, the penis slices were cultured with 3% H₂O₂ (10 min). The primary antibodies against collagen 3 (1:200, AF5457, Affinity, China), Smad7 (1:200, AF5147, Affinity, China), elastase-2B (1:200, orb471854, Biorbyt, China), and osteopontin (1:200, AF0227, Affinity, China) were cultured with slices overnight at 4 °C, respectively. After PBS washes, the goat anti-rabbit IgG (H + L) secondary antibody (1:5000, #S0001, Affinity, China) was cultured with slices at 37 °C for 60 min. After staining with diaminobenzidine (DAB), the slices were dehydrated, purified and sealed with neutral gum. The results were observed and photographed with a light microscope.

Based on a previously reported protocol [1 (link)], the dorsal neurovascular bundle and buck fascia were removed from the TA and urethra. The tissues and fibroblasts in each group were lysed in RIPA buffer (R0010, Solarbio, China). Twelve per cent SDS-PAGE was used to separate proteins (30 μg), and the separated proteins were transferred to PVDF membranes (EMD Millipore). Appropriate primary antibodies diluted with 5% BSA were incubated with the membranes overnight at 4 °C. The primary antibodies consisted of collagen 3 (1:800, AF5457, Affinity, China), Smad7 (1: 800, AF5147, Affinity, China), elastase-2B (1: 800, orb471854, Biorbyt, China), osteopontin (1: 800, AF0227, Affinity, China) and GAPDH (1:1000, BA2913, Boster, China). After 3 washes with TBS-0.01% Tween 20, the HRP-Affinipure goat anti-rabbit IgG (H + L) (1:500, BM3894, Boster, China) was incultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).

The heart tissues were fixed with 4% paraformaldehyde in phosphate buffered saline at room temperature for 48 hours and embedded in paraffin. The samples were cut into 5μm, and stained with hematoxylin/eosin (HE) to evaluate the cardiac histological morphology. Masson’s trichrome staining and Sirius red staining were used to measure cardiac collagen deposition. The slides were incubated overnight at 4°C with the specific primary antibodies against Collagen I (14696-1-AP, Proteintech), Collagen III (AF5457, Affinity), TAFAZZIN (sc365810, Santa Cruz Biotechnology), CRLS1(14845-1-AP, Proteintech) and OPA1(ab157457, Abcam) for immunohistochemistry.