Af0227

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).

This experiment was grouped into control, US, HN-Ti₃C₂, and HN-Ti₃C₂+US groups. hBMSCs seeded on glass coverslips in 24-well plates were incubated for 14 or 21 days under the corresponding conditions according to the groups. The US and HN-Ti₃C₂+US groups were treated with US (1.0 MHz, 0.2 W/cm², 50% duty cycle) every two days for 10 min each time, for a total of 4 times. Cells were washed and fixed with 4% paraformaldehyde for 30 min before permeabilized with 0.5%Triton x-100 for 15 min. After sealing with blocking solution for 30 min, alkaline phosphatase (ALP; rabbit source, DF6225) and OPN (Affinity, rabbit source, AF0227) antibodies diluted in the antibody diluent were added to the corresponding wells and incubated for 12 h at 4 °C. After discarding the antibody, the wells were washed three times with 0.1% PBST and then anti-rabbit antibody (red fluorescence) (Proteintech, SA00013-4) was added to each well and incubated in the dark for 1 h at room temperature. Anti-rabbit antibody was discarded, and the wells were washed again with 0.1% PBST before staining the cytoskeleton and nucleus. The cytoskeleton was stained with green fluorescence-labeled phalloidin solution (Yeasen, 40735ES75), and the nuclei were stained with DAPI solution (Beyotime, P0131). Fluorescence staining images were captured using a fluorescence microscope.