Anti cd31

For IHC, the liver tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (4-μm thickness). They were then dewaxed, hydrated for 30 min at room temperature, and incubated overnight at 4 °C with the following primary antibodies: anti-CD31 (1:1000, Proteintech, Wuhan, China); anti-CD206 (1:10000, Proteintech, Wuhan, China), anti-HECTD3 (1:400, BIOS, Beijing, China). Subsequently, the slides were incubated with a horseradish peroxidase-labeled secondary antibody, and the immunoreactivity was visualized with 3,3-diaminobenzidine tetrahydrochloride (DAB). Tissue sections were counterstained with hematoxylin and visualized using Leica Microsystems at 200 × and 400 × magnification. Image-pro plus 6.0 was used for image analysis (Media CybernetiHOPE, Inc., Rockville, MD, USA).
IF was performed as previously reported^{44 (link)}. Specifically, the sections or cell slides were incubated with anti-CD31, anti-CD206, anti-TRAF3 (1:100, Proteintech, Wuhan, China), anti-Ly6G (1:100, Novus, Littleton, USA), anti-MPO (1:200, Servicebio, Wuhan, China), anti-CD11b (1:500, Servicebio, Wuhan, China) and anti-HECTD3 (1:100, BIOS, Beijing, China) antibodies. After washing, the slides were incubated with Alexa Fluor 594-conjugated secondary antibodies or Alexa Fluor 488-conjugated secondary antibodies (1:200, Proteintech, Wuhan, China).

Penises were fixed with 4% formaldehyde supplemented with 0.002% picric acid for 4 hours at room temperature and were then dehydrated in 30% sucrose PBS overnight at 4 ^°C. Fixed tissues were embedded in optimal cutting temperature (OCT) compound (Leica) and cut into 10-μm sections. Sections were blocked with 0.1% Triton X-100/0.1% Tween 20/3% nonimmune goat serum in PBS for 1 hour at room temperature Sections were incubated with primary antibodies (anti-MYPT1 (1:200, Cat. No. 22117-1-AP, Proteintech); anti-smooth muscle α-actin (1:200, Cat. No. ab7817, Abcam); anti-eNOS (1:200, Cat. BS3571, BioRad); antidesmin (1:200, Cat. No. 21404-1-AP, Proteintech); anti-SMHHC (1:200, Cat. No. 16520-1-AP, Proteintech), anti-CD31 (1:200, Cat.No.550274, BD Biosciences) overnight at 4 ^°C and then with fluorescent secondary antibodies (Invitrogen) for 2 hours at room temperature. Fluorescence staining was examined under a confocal microscope (Olympus).

The animal tissues were embedded in paraffin and then processed for IHC. The paraffin sections were incubated with anti-SET8 (ProteinTech, Wuhan, China), anti-FOXO1 (Cell Signaling Technology, Danvers, MA), anti-PTEN (Proteintech, Wuhan, China), and anti-CD31 (Proteintech, Wuhan, China) antibodies overnight at 4 °C in a humidified chamber. Double staining was performed with the use of diaminobenzidine and fast red as the enzyme substrates according to the manufacturer’s instructions.