Worm Breeder's Gazette 11(1): 68
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.
Hoping they might eventually prove more sensitive than biotin- labelled probes and allow better localization than [35S]-labelled probes, I have been doing in situ hybridizations with digoxigenin- labelled probes. When I stained pure DNA or RNA fixed to slides, I saw punctate staining. The number but not the brightness of the points depended on concentration, suggesting each point was an individual molecule. (A molecule would have contained 1300 nucleotides of labelled DNA carrying 60 molecules of digoxigenin. From the number of points, each contained at most ten times that.) him- 8 squashes stained with labelled vit-2 DNA (made from the plasmid DJ1- B given me by Peg MacMorris) by the protocol below had punctate staining in adult hermaphrodite intestines, but not in other tissues or adult male intestines. There was no staining when a non-homologous probe was used. The only exception was a small amount of punctate staining on the surfaces of gonads with both vit-2 and nonhomologous probes. mab-3 mutations are known to cause yolk expression in males, and both male and hermaphrodite mab-3; es stained. The brightness of the points was the same as I saw with pure DNA or RNA, suggesting they might be individual mRNA molecules. If so, I detected <1% of the message: there were <100 points in an intestinal cell, where a rough calculation suggests there should be 10+E4-10+E5 molecules of vit-2 mRNA. I hope this dismal detection efficiency will be improved by optimizing probe, fixation, etc. [See Figure 1] The acetylation step reduces non-specific probe binding, and powdered milk reduces non-specific antibody binding. The hybridization conditions were worked out using pure RNA fixed to slides. Aside from these, it should not be assumed that there is a good reason for any step. Contact me for a current, excruciatingly detailed protocol.