Uniform Staining of Nerve Endings in Skeletal Muscle with Gold Chloride

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Abstract
Skeletal muscle, teased into strips several millimeters long and 1 mm or less thick, is fixed in a 3:1 mixture of lemon juice and formic acid until transparent. The tissue is compressed between folds of absorbent material to remove excess fixative and impregnated for 10 min in a volume of 1% gold chloride equal to the volume of muscle. After removing excess gold chloride, reduction is effected. Method a. Exposure to 25% formic acid in the dark for 6 hr. When intramuscular nerves are visible, the muscle is further teased with glass instruments. Reduction is continued for an additional 18 hr in 25% formic acid, and the tissue is cleared in glycerol for 24 hr. Method b. Exposure to intense artificial visible light while the tissue is just immersed in distilled water at 37° C. Colour develops in the nerve endings as in a photographic print. When nerves only are stained (approximately 0.5 hr), the lamp is removed and microdissection at room temperature commenced. When nerve endings in muscle spindles are just visible (approximately 1 hr), the distilled water is replaced by glycerol. After either method of reduction, areas of motor end-plates and individual sensory receptors are isolated by microdissection. Preparations are mounted in a small drop of glycerol, considerable pressure is applied to the cover slip, and the cover ringed with a sealing fluid.