Denervation and neuromuscular junction (NMJ) staining

For sciatic nerve denervation, mice were anesthetized with ketamine. The right hind limb was prepared for surgery, a 1-cm incision was made in the skin along with the axis of the femur, and the sciatic nerve was isolated. To prevent reinnervation, 3–5 mm section of the sciatic nerve was cut and removed. Mice were sacrificed by cervical dislocation 2 weeks after denervation. For NMJ staining, muscles were dissected and fixed by 4% PFA and incubated 30% sucrose/PBS for overnight. Muscles were frozen by cold isopentan with OCT-compound and sectioned longitudinally at 50 µm thickness. Sections were incubated with blocking buffer containing 0.1% Triton-X 100 and biotinylated α-bungarotoxin (α-BTX) to detect NMJs for overnight. NMJs were visualized by FITC-conjugated streptavidin.