Pkh2 green fluorescent cell linker kit

The obtained MNCs (5×10⁶ cells) were labeled with a green fluorescent marker using a labeling kit (PKH2 Green Fluorescent Cell Linker Kit; Sigma-Aldrich) and immediately transplanted to the left coccygeofemoral muscle of the same rabbit, as described previously (Figure 2A).14 (link) Briefly, the labeled MNCs were suspended in 1 mL phosphate buffered saline (PBS), and equal volumes were injected with a 25-gauge needle in the coccygeofemoral muscle at 3 different sites. The outline of the coccygeofemoral muscle was clearly identified through the moistened buttock skin. The 3 injection sites were set at equal intervals on the longitudinal median line of the muscle, and the needle tip was set at a depth of 10 mm from skin surface. At days 3 (n=3) and 7 (n=3) after transplantation, the coccygeofemoral muscle and the adductor muscle (a major muscle proximal to the coccygeofemoral muscle) were excised and divided transversely into 8 samples, which were frozen at −80°C. These frozen samples were cut in 4-μm–thick sections and analyzed using fluorescent microscopy.