A simple and rapid whole-mount staining method with methyl green-pyronin G applied to a molluscan brain preparation

Subesophageal ganglia of molluscs have been stained as whole mounts with methyl green-pyronin G to display the relative location of individual neurons. Nuclei appear blue, perikarya red. Expose the ganglion cells by dissection of the connective tissue in snail Ringer. Transfer the ganglion to a fixative of 2.5% glutaraldehyde in 0.1 M Na cacodylate pH 7.1 at 4 C for 12--24 hours, and wash in distilled water for 1 1/2 hours. Stain with methyl green-pyronin G for 1/2--1 hour and differentiate in 96% ethanol using many rapid changes. Transfer the ganglion to absolute ethanol for 2 1/2 hours and clear in xylene for 3 hours before embedding in Depex in a suitable dish. When the Depex has hardened, the preparation can be stored, and is readily available for subsequent examination. The method may be applicable to other invertebrate tissues, and may be useful in preparing objects for teaching purposes.