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The collected cavernous nerve and penis samples were fixed in 4% paraformaldehyde for 24 h at 4 °C before creating a paraffin block. The following primary antibodies were used: Neuronal nitric oxide synthase (nNOS, diluted 1:200; Santa Cruz Biotechnologies, Santa Cruz, CA, USA), alpha smooth muscle actin (α-SMA, diluted 1:500; Abcam, Cambridge, UK), vascular endothelial growth factor (VEGF; diluted 1:200; Santa Cruz Biotechnologies), basic fibroblast growth factor (bFGF, diluted 1:500; Cell Signaling Technology), stromal cell-derived factor-1 (SDF-1 diluted 1:200; Abcam), and platelet endothelial cell adhesion molecule (PECAM-1, diluted 1:500; Abcam), and 6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Inc., Burlingame, CA, USA) was used to stain nuclei. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2012 (Zeiss).

Animals were sacrificed by intraperitoneal injection with sodium pentobarbital (50 mg/kg), followed by bilateral thoracotomy. Penile midshaft tissues were freshly harvested and fixed [23 (link)], and immunofluorescence staining was performed as previously described [31 (link)]. The corpus cavernosum paraffin sections were immunostained with VEGF (diluted 1 : 200 Santa Cruz Biotechnologies, Santa Cruz, US), neuronal nitric oxide synthase (nNOS, diluted 1 : 200 Santa Cruz Biotechnologies, Santa Cruz, CA), brain-derived neurotrophic factor (BDNF, diluted 1 : 200; Abcam, Cambridge, UK), nerve growth factor (NGF, diluted 1 : 200; Abcam, Cambridge, UK), stromal cell-derived factor-1 (SDF-1 diluted 1 : 200; Abcam, Cambridge, UK), and neuron-specific β-III tubulin (diluted 1 : 200; Abcam, Cambridge, UK) and mounted with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA) to stain the nuclei. The collagen and vascular smooth muscle was evaluated after tissue staining with the Masson trichrome technique using the method previously described [32 (link)]. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2009 (Zeiss).

The sections were prepared as described above. For immunohistochemistry, sections were deparaffinized in xylene and dehydrated through a graded series of alcohol. High-pressure antigen retrieval was performed with citrate antigen repair solution and then incubated in 3% hydrogen peroxide at room temperature for 20 min. The slices were incubated with primary rabbit polyclonal antibodies against stromal cell-derived factor-1 (SDF-1, 1:200 dilution, Abcam, Cambridge, MA) at 4 °C overnight and then incubated with a horseradish peroxidase-labeled secondary antibody at 37 °C for 30 min. Next, 3,3′-diaminobenzidine (DAB) was added at room temperature for 10 min, and the slices were then stained with hematoxylin at room temperature for 2 min. Finally, the slices were gently washed with deionized water, dehydrated in gradient alcohol solutions, mounted with neutral balsam, and observed using an optical microscope. Anti-CD31 and anti-CD34 antibodies (1:200 dilution, Abcam, Cambridge, MA) were used for immunofluorescence. As for immunofluorescence, the slices were incubated with the abovementioned primary antibodies at 4 °C overnight and then incubated with a secondary antibody at 37 °C for 1 h. The slices were finally stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA) and observed under a Zeiss LSM 700 confocal fluorescence microscope (ZEISS, Germany).

Histological analyses were performed as previously described ^{11 (link)}. The collected hearts were fixed with 4% paraformaldehyde (PFA) and embedded in optimal cutting temperature compound for sectioning. Frozen Sects. (5 μm) of transverse heart slices were incubated with antibodies against PDGFRα, CD90, SDF-1, HGF, PGF, Ang1 (Abcam, Cambridge, UK) and SMA (Dako, Glostrup, Denmark). Samples were then probed with Alexa Fluor 555- or 488-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA). The frozen sections were also labeled with AlexaFluor 647-conjugated ILB4 (Invitrogen) following the manufacturer’s instructions. Nuclei were counterstained with Hoechst 33,342 (Dojindo, Kumamoto, Japan). The labeled sections were assessed using a Biorevo BZ-9000 (Keyence, Osaka, Japan). Finally, 5–10 fields were captured for each specimen, and the results were expressed as cell density. Transversely sliced heart sections were stained with Picro-Sirius Red and assessed by optical microscopy. Percent fibrosis was calculated as the percentage of pink-colored collagen in the total area.

To assess angiogenesis and SDF-1 expression in the infarct border zone, the heart tissue sections were deparaffinized, rehydrated, permeabilized, and then stained with conjugated antibodies against CD31 (Abcam), α-SMA (Abcam), or SDF-1 (Santa Cruz). Then, the sections were incubated with respective secondary antibodies conjugated with Alexa fluor 488 (Invitrogen) or color reaction with the DAB kit. The samples were analyzed using a confocal microscope (LSM 780, Zeiss). Positively stained cells were counted in three sections per heart, five high-power fields (HPFs) per section. Vascular density was quantified as the number of CD31+ cells per HPF. Arteriole density was quantified as the number of α-SMA+ cells per HPF. SDF-1 expression in peri-infarcted heart tissue at 1 day, 3 days, and 7 days after AMI was also quantitatively evaluated by rat SDF-1 ELISA kit (Elabscience, E-EL-R0922c) according to the manufacturer’s instructions.