Worm Breeder's Gazette 13(1): 24 (October 1, 1993)

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.

FISHing for complements

Phil Birchall, Donna Albertson

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, U.K.

Fluorescence in situ hybridisation (FISH) has been used to detect unc-54 mRNA in post-embryonic stages of N2 .

The technique is fast (same-day results) and produces a high resolution signal in about 90% of the sampled nematodes. All washes are done in an eppendorf tube; the worms are spun down, the old wash removed and replaced with 1ml of the next solution. The nematodes are fixed for 6 hours in 3.7% formaldehyde in 0.1M HEPES-KOH (pH 7.5) buffer, permeabilised in 5%-ß-mercaptoethanol and 50µg/ml Proteinase K and taken into hybridisation buffer (40% formamide, 4xSSC, 0.1M HEPES-KOH (pH 7.0), 80µg/ml sheared salmon sperm DNA, 1.6% SDS and 0.024% SLS). Probes are prepared by one of four methods; PCR, in vitro transcription, nick translation or random priming. In each case, a fluorochrome-conjugated nucleotide is incorporated into the sequence during the polymerase-catalyzed reaction. Hybridization is at 45°C for at least four hours. The nematodes are washed, mounted on a slide, mixed with DABCO (retards fading of the fluorescent signal) and viewed. There is very little background at all (negative controls are hard to find on a slide) and the hybridization signal can be seen with a conventional fluorescence microscope.

The nematodes have been simultaneously hybridized with a 1kb ribosomal DNA segment labeled with rhodamine-4-dUTP (Amersham), and with a 1kb unc-54 sequence labeled with fluorescein-12-dUTP (Boehringer Mannheim). In this case, the nucleoli of all the cells appear bright red, the cytoplasm of all the cells appears faintly red and the cytoplasm of the body wall muscle cells appears green, in animals from L1 to adult. These signals are sensitive to RNaseA. An unc-54 -specificsignal is also seen in the vulval and uterine muscles at the L4 /youngadult stage. Embryos are currently being examined.

We are extending our investigations to include other genes to gauge the sensitivity and generality of the technique.