An Acetylcholinesterase Method for In Toto Staining of Peripheral Nerves

Abstract

Stomach, small intestine, uterus, urinary bladder, vagina, mesentery, mesometrium and joint capsule of rats, gall bladder, cystic duct and bile duct of dogs and uteri of young children are stained in toto. Procedure: Tissue is perfused with saline containing hyaluronidase, then pinned on a flat layer of Paraplast and fixed for 24 hr in cold sucrose formol solution. Stomach, urinary bladder and gall bladder are also fixed in toto. Rinse for 2 days in cold 0.22 M sucrose in a sodium cacodylate bnffer pH 7.2. Incubate in medium consisting of 60 mM acetate-buffer pH 5.0 M pH 5.6 (for human material only), 2 mM acetylthiocholine iodide, 15 mM Na citrate, 3 mM Cu sulphate, 0.5 mM K₃Fe(CN)₆, 5 × 10⁻⁴ M iso-OMPA, 1% Triton × 100 at 37°C. Rinse in doubly distilled water. Dehydrate in glycerine/water mixtures of increasing glycerine content. Store in glycerine or delaminate under dissecting microscope. Delaminated specimens are mounted on gelatinized object glasses, cleared in xylene and coverslipped with Malinol. Specimens stored in glycerine can be studied microscopically. Stained specimens can also be embedded in Paraplast and sections can be studied after counterstaining.