Immunofluorescence was used to determine the smooth muscle content in the cavernosum and nNOS content in the dorsal penile nerve. Penile tissue sections (5μm) were dewaxed in dimethylbenzene and then washed in phosphate buffered saline (PBS) three times. The slices were blocked by normal goat serum at room temperature for 1h and incubated with the antibody against α-smooth muscle actin (α-SMA; 1:100, Boster), nNOS (1:200, BD Biosciences) or neurofilament (1:200, ProteinTech) at 4°C overnight in a humidified and lightproof chamber. After three washes in PBS, the slices were incubated with DyLight-conjugated secondary antibodies (1:200, Abbkine, Redlands, CA, USA) for 1h at room temperature. Nuclei were stained with DAPI (Beyotime, Shanghai, China).
Masson’s trichrome staining was performed to assess the degree of cavernous fibrosis, which was determined by the ratio of collagen to smooth muscle in penile tissues of all groups.
Images were all obtained using fluorescence microscopy (Olympus, Tokyo, Japan) and analyzed by Image-Pro plus software (Media Cybernetics).
Masson’s trichrome staining was performed to assess the degree of cavernous fibrosis, which was determined by the ratio of collagen to smooth muscle in penile tissues of all groups.
Images were all obtained using fluorescence microscopy (Olympus, Tokyo, Japan) and analyzed by Image-Pro plus software (Media Cybernetics).