Pecam 1

Cells were washed with cold PBS and proteins extracted with Laemmli's buffer. Samples were run on 10 or 12% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with StartingBlock buffer (ThermoScientific) and probed with primary antibodies overnight at 4°C: VEGFR3 (R&D systems), phospho-VEGFR3 (Cell Applications), VEGFR2 (Cell Signaling), PECAM-1 (Abcam), VE-cadherin (Santa Cruz), GFP (Invitrogen) and actin (Santa Cruz). DyLight conjugated fluorescent secondary antibodies (680 nm and 800 nm, Thermoscientific) or HRP-conjugated antibodies were used to detect primary antibodies. Bands were detected and quantified with an Odyssey infrared imaging system for DyLight antibodies (Li-Cor) or a BioRad western blot imaging system (Bio Rad).

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).

Tumor samples were collected from mouse xenografts and fixed in 10% neutral-buffered formalin (Sigma-Aldrich). Slides were then stained using H&E (Sigma-Aldrich) according to the manufacturer's instructions. For immunoblotting, protein lysates were prepared by homogenizing the frozen tumor tissues. Protein quantitation and immunoblotting were then performed as described above with 30-μg protein/sample. For immunohistochemistry, paraffin-embedded ovarian tumor tissues were serially sectioned into 5-μm slices. The prepared slides were then deparaffinized, rehydrated in xylene and graded alcohols, and then rinsed in PBS. The slides were also incubated in 5% hydrogen peroxide in methanol for 20 min to block endogenous peroxidase activity. Sections were incubated with a saturating concentration of anti-mouse CD31 (platelet-derived endothelial cell adhesion molecule; PECAM-1) (Abcam) antibody overnight at 4°C, followed by a streptavidin-peroxidase complex at room temperature for 1 h. Microvessel density (MVD) was quantified in five randomly selected individual tumor fields (at 40× magnification) per sample, and the number of microvessels was counted under a high-powered microscope (400× magnification). All immunochemical analyses were performed using an Axiophot 2 apparatus (Carl Zeiss MicroImaging Inc., Thornwood, NY).

In order to examine the survival and differentiation of N-ADSCs in the denervated muscles, we anesthetized the mice and performed the perfusion fixation on day 28. Then, the muscles were removed and histological and immunohistochemical assays performed. The frozen slices were subjected to immunofluorescent and immunohistochemistry staining. The muscle slices were stained with CD31 (PECAM-1; Abcam) and the nuclei with DAPI (Dako) as described previously [7 (link)].

Streptozocin (STZ), insulin for cell culture, and MGO were from Sigma-Aldrich (St. Louis, MO, United States). ProLong gold antifade mounting reagent with DAPI was purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). RPMI 1640, DMEM, and phosphate buffer solution (PBS) were obtained from GIBCO (Carlsbad, CA, United States). Chemiluminescence reagent was from Millipore (Billerica, MA, United States). Insulin was from Roche chemical (Houston, TX, United States). OxiSelect MGO Competitive ELISA Kit was purchased from Cell Biolabs (San Diego, CA, United States). Matrigel was from BD Biosciences (Waltham, MA, United States).
The following antibodies were obtained from various suppliers: PECAM-1, collagen I, and collagen III were from Abcam (Cambridge Science Park, Cambridge); CBP, GLUT-1, VEGF-A, HIF-1α, and HRP-labeled secondary antibody were from Cell Signaling Technology (Danvers, MA, United States); and donkey anti-rabbit Alexa Fluor 488 was purchased from Thermo Fisher Scientific (Carlsbad, CA, United States).

Immunohistochemistry was performed by employing the avidin–biotin complex (ABC) method as has been described previously^{6 (link), 61} . Briefly, FFPE sections were deparaffinized, rehydrated and then microwaved for 30 min in citrate buffer (pH 6.0). Sections were incubated for 5 min with 3%H₂O₂ in methanol and then blocked for 1 h at RT with PBS containing 5% normal goat serum and 0.1% Triton X-100. Thereafter, they were incubated for 2 h at RT with primary antibodies. The primary antibodies used in this study included the rabbit anti-platelet endothelial cell adhesion molecules-1 (PECAM-1; 1:400) and rabbit anti-von Willebrand factor (vWF; 1:400, all obtained from Abcam, Cambridge, UK). After being washed, sections were incubated for 45 min at RT with biotinylated secondary antibody (1:200; Vector Laboratories, CA, USA), followed by peroxidise coupled ABC (Thermo Fischer Scientific, Waltham, MA, USA). Antibody binding was visualized using DAB-H₂O₂ for 5 min at RT followed by being counterstained with hematoxylin. They were then dehydrated, mounted with coverslips and observed under a light microscope. Scoring of immunolabeling positive cells for each antibody in different anatomical regions was done according to the method previously described^{6 (link)}.