Worm Breeder's Gazette 12(3): 12 (June 15, 1992)
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.
We have developed a method to histochemically stain C. elegans that does not kill the embryos inside the stained mother and we have used this method to isolate mutations that alter gene expression patterns (see the accompanying article by Guofeng Xie et al. on the selection of mutants based on histochemical staining patterns). To make C. elegans permeable to the histochemical stains, we modified Finney and Ruvkin's method to make C. elegans permeable for antibody staining. We have stained animals permeabilized in this way for esterase, ß-glucuronidase and , ß-galactosidase. This procedure should work for other histochemical stains as well. In our hands, worms made permeable by this method and stained for , ß-galactosidase activity are better stained than those made permeable by the freeze-dry method and stained for , ß-galactosidase. Essentially all animals taken en mass through this procedure are stained. Approximately 75% of the embryos inside the stained animals survived and there were an average of 14 + 8 (n= 17) surviving offspring from each stained mother. Our protocol varies slightly depending on whether we are staining for esterase or ß-galactosidase. We present the ß-galactosidase protocol here.
1. Formaldehyde preparation: mix 200 mg of paraformaldehyde with 900 µl of 5.0 mM NaOH. Heat the
solution at 65°C for 15 min. Centrifuge this mixture in a microfuge at 14,000 rpm for 1 min. Use the
supernatant immediately.
2. Wash healthy gravid worms from a 60 mm NGM plate with 1-2 ml of M9 and transfer the worms to a microfuge tube.
3. Let the worms settle for 5 min at room temperature and remove the supernatant.
4. Add 500 /µl of ice-cold 2X MRWB (160 mM KCl, 40 mM NaCl, 20 mM N a2 EGTA,10 mM spermidine HCl,
30 mM PIPES, pH 7.4 and 50% methanol), 100 µl of formaldehyde and 400 µl of water and mix well.
Incubate the tube for 35 min at 4°C. Occasionally mix the tube contents during fixation.
5. Wash 2X with 1.0 ml TTB (100 mM Tris HCl pH 7.4, 1% Triton X-100 and 1.0 mM EDTA).
6. Add 960 µl of TTB, 30 µl formaldehyde and 10 µl of ,ß-mercaptoethanol.
7. Place the tube on a rotator set at 2 rpm for 10 min at room temperature and then let the worms
settle for 5 min at room temperature. At this point the worms are fragile so treat them carefully.
8. Wash once with 1X B03 (40X B03 contains 1.0 M H3B03 and 0.5 M NaOH)
9. Add 900 µl of 1X B03 and 100 µl of 100 mM DTT.
10. Mix on a rotator at 2 rpm for 15 min at room temperature.
11. Wash twice with ddH20 letting the worms settle for 5 min each time.
12. Stain for ß-galactosidase activity. We use a very slightly modified version of Andy Fire's staining mix: 620 µl H20 ,250 µl 0.8 M Na phosphate (pH 7.5), 2 µl 1.0 M M gCl2 ,4 /ll 1% SDS, 100 µl Redox buffer (50 mM each of Potassium Ferricyanide and Potassium Ferrocyanide), 5 µl 5.0 mg/ml Kanamycin Sulfate and 20 µl 2% X-GAL in Dimethylformamide.
13. We stain at room temperature for 8-10 hours (but the staining time will vary with the lacZ construct).