NO gel solution (200 μl) with P6 hMSCs (106 cells/ml; n = 2) or without cells (hMSC-free control) was injected into two subcutaneous sites (right and left symmetrically) of 6-week-old male BALB/c nude mice and maintained for 3 weeks. To visualize perfusable vessel formation in the plug sites, mice were perfused with a heparinized fluorescent microbead solution (Thermo Fisher Scientific). Briefly, heparinized saline (10 IU/ml) was injected into the left ventricle after amputating the internal vena cava, followed by perfusion of heparinized fluorescent microbead solution in saline. Gel plugs were then harvested and subjected to LSM780 confocal imaging. The paraffin sections were obtained from the plug parts and stained with MT, as previously described in the above sections. To confirm neovascularization into gel plugs, tissue section slices were stained with primary rabbit anti-Flk1 antibody (Santa Cruz Biotechnology) and secondary phycoerythrin (PE)–conjugated goat anti-Rabbit IgG (Thermo Fisher Scientific) antibody, followed by LSM780 confocal imaging.

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