S0011

Rats were perfused transcardially with 0.1 M phosphate-buffered saline (PBS), and the spinal cord (L4-6) tissue was fixed in 4% paraformaldehyde. After 24 h, the fluid was changed to 20% sucrose in 0.1 M PBS, and then the fluid was changed to 30% sucrose in 0.1 M PBS for 24 h to achieve extensive dehydration. The spinal cord specimens with a thickness of 14 μm were prepared by a freezing microtome (Leica, Germany). After blocking with 5% non-fatty dry milk for 1 h at room temperature, the sections were incubated with primary to rabbit anti-SIRT2 (1: 400, ab51023, Abcam) or rabbit anti-FPN1 (1:500, NBP1-21502, Novus), and either mouse anti-NeuN antibody (a neuronal marker, 1:400, ab104224, Abcam) or mouse anti-CD11b (1:400,CD11b, 1:400, MCA275R, BIO-RAD) overnight at 4°C, followed by the administration of Alexa Fluor 488 goat anti-mouse secondary antibody (1:2,000, Thermo Fisher Scientific, United States), and goat anti-rabbit IgG (H+L) CY3-conjugated antibody (1:400, S0011, Affinity Bioscience, Jiangsu, China). The double-stained sections were captured by a confocal laser-scanning microscope (Leica, Germany).

According to the manufacturer's protocol, superoxide anion and mitochondrial superoxide in the corpus cavernosum were detected with dihydroethidium (Beyotime Biotechnology, China) and MitoSOX (Invitrogen, USA). DAPI was used to stain the cell nuclei.
For immunofluorescence staining, penile sections were incubated with primary antibodies against SMA (1 : 200, Affinity Biosciences, AF1032), GPX4 (1 : 200, ABclonal, A1933), and ACSL4 (1 : 300, Affinity Biosciences, DF12141). Secondary antibodies included DyLight 488- (1 : 300, Affinity Biosciences, S0008), 594- (1 : 300, Affinity Biosciences, S0006), and CY3- (1 : 300, Affinity Biosciences, S0011) conjugated antibodies. Nuclei were stained with DAPI. Images were acquired using a fluorescence microscope (Nikon, Japan) or a confocal laser scanning microscope (Zeiss LSM 880, Germany).
Masson's trichrome staining was performed according to the standard protocol [30 (link)]. The ratio between smooth muscle and collagen in the corpus cavernosum reflects fibrosis of the corpus cavernosum. ImageJ 1.46 (National Institutes of Health, USA) was used for quantitative analysis of the images.