Acetylcholinesterase (AChE) demonstrated by a vascular perfusion incubation technique

Abstract

For the cytochemical demonstration of enzymes, the commonly employed procedures involve using chopped small cubes or sections of tissue incubated in a proper medium. With the chopped tissue, penetration of substrate and capturing agents is very slow. When using frozen sections artifacts are difficult to avoid. Also proper vibratome sections are hard to achieve with small organs such as the pineal gland. In order to overcome these disadvantages, we have developed a vascular perfusion incubation technique for the demonstration of AChE.With this technique, a simple gravity flow apparatus for vascular perfusion with two glass containers for fixative and Tyrode solution was used. A needle (19g) with a 32 cm long vinyl tube was connected to the apparatus by a three-way valve. Adult Djungarian hamsters were anesthetized with 1.5 g/kg urethane. The needle was inserted into the ascending aorta through the left ventricle. The following solutions were then perfused for the times indicated: Tyrode, 1 min; fixative (1% purified glutaraldehyde and 2% formaldehyde, 0.05 M cacodylate buffer, pH 7.2), 5 min; Tyrode, 10 min; substrate-free incubation medium, 10 min; complete incubation medium, 20 min; pause for 10 min; repeat of the last two steps four times; isotonic sodium sulphate, 10 min; buffered sulphide, 30 min; isotonic sodium sulphate, 10 min; fixative (3% glutaraldehyde and 2% formaldehyde, 0.05 M cacodylate buffer), 5 min. The entire pineal gland and pieces of the tongue were taken. After osmium postfixation standard electron microscopic procedures were used.