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

Abstract

Longitudinal 50-100 mum-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 NaSO3 (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 muscle 3 1/2-4 hours after receipt of a sample, and makes possible determination of the terminal innervation ratio.