Worm Breeder's Gazette 7(1): 72
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
I have used the following procedure to stain C. elegans embryos histochemically for AChE activity. 1. On a microscope slide subbed with a 1% BSA solution, place a few young adults into a 20 l drop of 2% glutaraldehyde solution in 100 mM maleate buffer, pH 6.0. The animals are cut with a scalpel blade at the vulva to release the embryos. 2. Very gently, a 16 X 16 mm coverslip is placed on top of the buffer drop. The slide is then immersed into a liquid nitrogen container for about 15 seconds, and the coverslip is quickly pried off with a scalpel blade. 3. Another 16 X 16 mm coverslip with its two opposite edges coated with grease is placed over the embryos. The slide is incubated in a humid chamber at O C for two hours. 4. About 10 l of the staining solution ( containing 10 mg acetylthiocholinechloride, 8.8 ml 100 mM maleate buffer, pH 6.0. 1ml 50 mM sodium citrate, 100 l 300 mM copper sulfate, and 100 l potassium ferrocyanide, added in this order) is applied at the open edge of the coverslip, and is absorbed at the other open edge with an absorbant paper ( see fig.). This step is repeated 3-4 times to ensure that all embryos have been immersed in the staining solution. The slide is then incubated in a humid chamber at room temperature for about 6 hours before light microscopy. A reddish-brown stain appears in the regions showing AChE activity. [See Figure 1] The above procedure can also be used for larvae and the adult animals. Significantly, this method reveals the same areas of AChE activity in C. elegans as in the method reported by Culotti et al. ( Genetics: 97, 281-305,1981), in which the animals are treated with 95% acetone for 3 minutes. The prominent areas showing the stain for AChE activity are, the nerve ring, lateral ganglion, ventral nerve cord, dorsal nerve cord, pre-anal ganglion, and the pharryngeo-intestinal valve cells.