A Cholinesterase-Bielschowsky Staining Method for Mammalian Motor End Plates

Abstract

Specimens of fresh mammalian muscle not more than 5 mm thick were fixed for 6 hr at 20-25 C in 10% formal-saline, washed in distilled water for at least 16 hr and sectioned in 10% sucrose at 50 μ with a freezing microtome. The sections were washed in distilled water for 1 hr and incubated at 37 C for 15 min in: 0.1 M CuSO₄, 0.2 ml; 0.5 M amino-acetic acid, 0.2 ml; 0.1 M acetate buffer, pH 4.7, 5 ml; and 0.8 ml of the supernatant fluid obtained from centrifuging a mixture of 2% acetylthiocholine iodide, 3 volumes, and 0.1 M CuSO₄, 1 volume. After incubation, they were washed in distilled water for 5 min, and placed in 5% ammonium sulphide for 2 min, washed in distilled water for 1 hr and placed in 10% neutral formalin and 2% pyridine in normal saline for at least 15 days. After formalin-pyridine, they were washed in tap water for 1 hr and stained in 10% AgNO₃ for 1.5 hr at 37 C in the dark, then washed in 10% formalin in tap water with changes until the white precipitate disappeared. Next they were warmed in 10 ml of ammoniacal AgNO₃ (3 excess drops of ammonia added) for about 1 min and, in turn, rinsed in 1% ammoniated water for 2 min, 1% acetic water for 2 min, distilled water for 2 min, 3% Na₂S₂O₃ for 5 min, and distilled water for 30 min; then dehydrated, and mounted in balsam. The motor end plates were seen as black deposits of Ag₂S. The nerve fibres showed the usual appearance following a silver stain and could be traced to their termination in the motor end plates.