Worm Breeder's Gazette 11(2): 20a
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.
Various protocols for permeabilization and staining of adult C. elegans in such a way as to avoid killing embryos in the uterus have been described (Sebastiano et al Genetics 112 459, AF WBG 9#2, Stone and Shaw WBG 10#3 p27, Candido MRC lore). I have been trying to optimize this procedure to obtain strong, well localized staining of lines carrying -gal fusion proteins, to allow for selection of altered staining patterns after mutagenesis. A current protocol is described below. The treatment is somewhat more drastic than previous methods, but allows excellent staining and reasonable preservation with survival of 3-5 embryos per stained adult. 1. Pellet animals from H20 and resuspend in 50% acetone for 10-15 min at room temp (the time for this step is critical...too long and embryos are inviable, too short and the adults fail to permeabilize and awaken in the staining solution.). 2. Wash animals 3x in PMB and resuspend in staining mix. Stain at room temperature. The hatched animals can survive within the adult in staining solution for several hours, but eventually die or free themselves, limiting staining time to about 12 hr. 3. Scoring with a dissecting microscope is best with epi-illumination over a white background PMB: 50mM NaPi pH 7.5, 1mM MgCl2 Staining Mix: PMB + 10mM KFerrocyanide + 10mM KFerricyanide + .004% SDS + 10 l/ml of 12.5% XGAL in DMF (XGAL stock should be stored at - 70 C) Optimal permeabilization so far has been around 95% of animals. Thus the technique could be used to screen for ectopic expression or expression of an otherwise inactive construct, but loss of expression would be difficult to screen for. N.B. Some permeabilization of pharyngeal muscle occurs without the acetone step (putting animals directly into staining solution).