Ab76067

Four weeks after the ASC or saline injection, the rats were sacrificed using an overdose of Zoletil 50. Skin from the right hind paw was excised, fixed in formalin, embedded in paraffin and 4-μm-thick specimens were mounted on saline-coated slides, deparaffinized and rehydrated in a series of graded alcohol solutions. This was followed by antigen retrieval in citrate buffer (pH 6.0; 0.1 mol/L), heating to 121 °C for 10 min and cooling to room temperature. To quench the endogenous peroxidase activity, the sections were further incubated for 5 min in 3% H₂O₂. Non-specific binding was blocked by incubation with 5% goat serum in PBS for 30 min. The blocked sections were incubated overnight at 4 °C with rabbit polyclonal antibodies against cyclooxygenase (COX)-2 (#12282, 1:200 dilution; Cell Signaling, Danvers, MA, USA), iNOS (ab15323, 1:200; Abcam, Cambridge, MA, USA) and nNOS (ab76067, 1:200; Abcam), rinsed and incubated with horseradish peroxidase-conjugated secondary antibodies for 30 min at room temperature. The slides were exposed to the colorimetric reagent 3,3-diaminobenzidine for 5 min, counterstained for 1 min with Mayer’s hematoxylin and mounted for evaluation.

Twenty-four hours after drug administration, the mice were anesthetized with pentobarbital sodium (80 mg/kg, i.p.) and euthanized by decapitation. The bilateral mPFC regions were dissected immediately and homogenized in RIPA Lysis Buffer (Beyotime, P0013B) with 1 mM PMSF (Sigma, P7626), and dissociated by sonic disruption. The homogenate was centrifuged at 14,000 g for 15 min, and the total protein concentration was assessed. The primary and secondary antibodies used in the Western blotting assay were anti-nNOS (1:500, Abcam, ab76067), anti-iNOS (1:500, Abcam, ab15323), anti-BDNF (1:1,000, Abcam, ab108319), anti-β-tubulin (1:1,000, Cell Signaling Technology, 2128), and goat anti-rabbit IgG (1:5,000, Abcam, ab6721) antibodies. An enhanced chemiluminescence (ECL, Thermo, 32109) reaction solution was added. The samples were then recorded on X-ray film (Carestream, 8294985) for visualization, and the immune reactivity was quantified using ImageJ software.

To determine the expression of C-kit and neuronal nitric oxide synthase (nNOS) in the stomach and small intestine, the stomach and small intestine were prepared in RIPA lysis buffer. The proteins were separated by 7.5% polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% skim milk for 1 h at room temperature, the membranes were incubated with primary antibodies, such as C-kit (0.1 μg/ml, AF1356, R&D Systems), nNOS (1:1,000, ab76067, Abcam) or α-tubulin (1:1,000, ab7291, Abcam) antibodies, overnight at 4°C. After washing with 0.1% TBS-T, the membranes were incubated with HRP-conjugated anti-goat (against C-kit, 1:2,500), anti-rabbit (against nNOS, 1:5,000), or anti-mouse (against α-tubulin, 1: 5,000) antibodies. These proteins were visualized using an enhanced chemiluminescence (ECL) advanced kit (Thermo Fisher Scientific, United States) and imaged using a FUSION Solo System (Vilber Lourmat, France). Protein expression was semiquantified using ImageJ (NIH).

Brain tissue from the right hemisphere was obtained, and proteins were extracted on the first day after I/R. The protein concentration of the samples was determined by the BCA protein detection Kit. In addition, 30 μg of protein from each group were loaded onto an 7.5 or 10% SDS–PAGE gel. After electrophoresis, the brain proteins were transferred to polyvinylidene fluoride (PVDF) membranes, blocked with 5% skim milk at room temperature for 1 h, and then incubated with the primary antibody overnight in a 4°C refrigerator. After an incubation with goat anti-mouse and anti-rabbit (Abmart, M21003, 1:2,000) secondary antibodies for 45 min at room temperature, membranes were washed with TBST, and then incubated with enhanced chemiluminescence (ECL) reagent (biosharp, BL520A, China) for detection. Primary antibodies included: anti-iNOS (1:1,000, 22226-1-AP, Proteintech, United States), anti-nNOS (1:1,000, ab76067, Abcam, United States, United States), and anti-eNOS (1:1,000, ab199956, Abcam, United States). β-Tubulin (1:1,000, 10094-1-AP, Proteintech, United States) was used for internal comparison. ImageJ software was used to quantitatively analyze the gray values of all protein bands.

Hippocampal tissues were washed by ice-cold PBS and lysed in RIPA buffer, followed by homogenizing mechanically. The homogenates were centrifuged at 12000 rpm for 15 min at 4°C. After electrophoresis on SDS-PAGE gels, proteins were transferred to membranes and blocked with 5% non-fat milk. Then, the blots were incubated with rabbit-anti-rat arginase-1 (ARG1) antibody (93668S, 1:1000; CST, USA), neuronal nitric oxide synthase (NOS1) antibody (ab76067, 1:1000; Abcam, UK), inducible nitric oxide synthase (NOS2) antibody (ABP51974, 1:1000; Abbkine, China), endothelial nitric oxide synthase (NOS3) antibody (#32027, 1:1000; CST, USA) and mouse-anti-rat β-actin antibody (66009-1-Ig, 1:5000; Proteintech, USA) at room temperature. After washing with PBST for three times, the blots were incubated with HRP goat-anti-rabbit IgG (SA00001-2, 1:6000; Proteintech, USA) and HRP goat-anti-mouse IgG (SA00001-1, 1:5000; Proteintech, USA) for 90 min. The color reaction was performed by ECL reagents. The band density was visualized after exposure to x-ray film and analyzed using the quantity one software (Bio-Rad, USA).

For in vivo cell tracking, paraffin-embedded cavernous nerve sections were immunostained with the following primary antibodies: cell nuclei (DAPI; Vector Laboratories, Burlingame, CA, USA) and anti-β-tubulin III. The penile tissue was sectioned to a thickness of 4 μm for staining with Masson trichrome for smooth muscle and collagen. The paraffin sections were used for immunostaining analysis, and the following primary antibodies were applied: anti-neuronal nitric oxide synthase (nNOS, diluted 1:100, ab76067; Abcam, Cambridge, UK), anti-β-tubulin III, anti-α-smooth muscle actin (α-SMA, diluted 1:250, ab5694; Abcam, Cambridge, UK), anti-endothelial nitric oxide synthase (eNOS, diluted 1:100, ab5589; Abcam), anti-CD31 (diluted 1:50, ab28364; Abcam), and DAPI. Using a microscope (Zeiss, LSM 510 Meta Confocal, Oberkochen, Germany), fluorescent images were captured, and the mean fluorescent intensity was analyzed using ZEN2012 software (Zeiss, Germany). An optical microscope (Olympus, BX50, Tokyo, Japan) was used to obtain digital images, and GraphPad Prism v5 software (GraphPad Prism Software, San Diego, CA, USA) was used to calculate the mean fluorescent intensity.

The penis was fixed using perfusion with 4% paraformaldehyde. It was then embedded in paraffin and sliced into 5-μm cross sections that were deparaffinized, rehydrated, and subjected to the Elastica van Gieson stain. Elastica van Gieson stain was performed by a standard method. In brief, the specimens were consecutively stained with resorcin‐fuchsin staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan), hematoxylin staining solution, and van Gieson staining solution (Muto Pure Chemicals, CO., LTD., Tokyo, Japan). For immunohistochemistry, sections were incubated with a primary antibody reactive to neural nitric oxide synthase (nNOS: 1:400, Abcam, ab76067) and heterochromatin protein-1γ (HP-1γ: 1:400, Abcam, ab66617). Sections were subsequently incubated with biotinylated secondary antibody and finally horseradish peroxidase-labeled streptavidin according to the instructions provided by the manufacturer (DAKO, Cambridgeshire, UK).