Ds qi2 digital

On E20, the injected fetuses were harvested, and a fluorescence stereomicroscope (M165FC, Leica, Germany) fitted with a Nikon DS-Qi2 digital camera (NY-1S35, Nikon, Japan) was used to take the back images of the fetuses. The skin lesion area of these fetuses was measured using BR analysis software. Then, the fetuses were perfused transcardially with 15 mL physiological saline, followed by 25 mL 4% paraformaldehyde. The lumbosacral spinal column—containing muscle, spinal cord, and subcutaneous tissue—was dissected from the fetuses and maintained overnight in 4% paraformaldehyde. The tissues were then dehydrated in 20% sucrose for 24 h and then embedded in the optimal cutting temperature compound (OCT, SAKURA, Japan). Serial 30-μm-thick slices from the lumbosacral spinal column were prepared using a freezing microtome (Microm HM525, Thermo, Germany) before attaching to polylysine-coated glass slides. The GFP-positive cells on the slides were observed and counted by fluorescence microscopy (80i, Nikon). All sections were marked and preserved at − 80 °C for TdT-mediated dUTP Nick-End Labeling (TUNEL) and immunofluorescence staining.