A Rapid Method for Demonstrating Skeletal Muscle Motor Innervation in Frozen Sections

Longitudinal 50-100 μm-thick frozen sections of muscle are picked up on slides coated with 3% EDTA and after drying are incubated to demonstrate acetylcholinesterase. Subsequent incubation in 0.5% K₃Fe(CN)₆ is followed by fixation for 30 minutes in formol-calcium or formol-saline. After washing, the slides are incubated in 20% aqueous AgNO₃ containing 0.1% CuSO₄ for 2-30 minutes at 37 C. Following development in a 1% solution of quinol (w/v) 5% with respect to Na₂SO₃ (w/v), axons and subneural apparatus stain dark brown to black in contrast to the less well stained muscle fibers and nuclei. This procedure permits study of the pattern of neuromuscular innervation in skeletal musk 31/2-4 hours after receipt of a sample, and makes possible determination of the terminal innervation ratio.