Worm Breeder's Gazette 16(2): 26

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.

Protocol for Antibody Staining Mitotic and Meiotic Chromosomes in the Gonad

Annette Chan, Barbara J. Meyer

HHMI and Department of Molecular and Cell Biology, U.C. Berkeley, Berkeley, California 97420-3204

We have devised an antibody staining protocol that is particularly useful for staining mitotic and meiotic chromosomes in the gonad. This procedure involves fixing and antibody staining dissected worms on a slide. The protocol preserves chromosome structure and produces bright antibody staining.

1. For any specific antibody try 1%, 1.5%, or 2% paraformaldehyde (PFA) with 5-minute fix:

a. Place worms in 5 m l 1X sperm salts on a positively-charged glass slide.

b. Using a needle (Precision Glide® Needle, Becton Dickinson & Co., 22 G 1 1/2), make an incision between the pharynx and gonad (closer to the gonad than the pharynx) or between the tip of the tail and gonad of each worm. To release the embryos, cut at the vulva.

c. Add 5 m l of 2%, 3%, or 4% PFA (Electron Microscopy Sciences, cat. #15710) in 1X sperm salts.

To prepare 2% PFA: 125 m l 16% PFA, 200 m l 5X sperm salts, 675 m l water

To prepare 3% PFA: 187.5 m l 16% PFA, 200 m l 5X sperm salts, 612.5 m l water

To prepare 4% PFA: 250 m l 16% PFA, 200 m l 5X sperm salts, 550 m l water

d. Place the slide in a humid chamber for 5 minutes. Place an 18-mm x 18- mm coverslip on top of the worms. Freeze the slide on a piece of dry ice. Let the slide sit on the dry ice for at least 10 minutes.

2. Remove the slide from the dry ice and quickly pop off the coverslip with a quick downward stroke of a single-edged razor.

3. Immediately place the slide in 95% ethanol for 1 minute. Be careful or the worms will fall off.

4. Incubate the slide in a vessel containing PBST for at least 10 minutes. Repeat this step twice.

5. Wick off most of the PBST, but do not allow the worms to dry. Add 30 m l primary antibody diluted in PBST. Carefully cover the worms with a 20 x 20-mm piece of parafilm. Place the slide in a humid chamber. Incubate overnight at room temperature.

6. Incubate the slide in a vessel containing PBST for at least 10 minutes. Carefully remove the piece of parafilm.

7. Incubate the slide in a vessel containing fresh PBST for at least 10 minutes. Repeat this step once.

8. Wick off most of the PBST, but do not allow the worms to dry. Add 25 m l secondary antibody diluted in PBST. Carefully cover the worms with a 20 x 20-mm piece of parafilm. Place the slide in a humid chamber. Incubate overnight at room temperature. Keep the slides in the dark whenever possible.

9. Incubate the slide in a vessel containing PBST for at least 10 minutes. Carefully remove the piece of parafilm.

10. Incubate the slide in a vessel containing fresh PBST for at least 10 minutes. Repeat this step once.

11. Wick off most of the PBST, but do not allow the worms to dry. Add 10 m l of 1 m g/ml DAPI or propidium iodide to the slide.

12. Mount the slide with 20 m l DABCO and a 22 x 40-mm cover glass. Seal the slide with fingernail polish.

13. Observe the slide. Store the slides at -20oC. Keep the slides in the dark whenever possible.

 

Sperm Salts: 50 mM PIPES, pH 7.0, 25 mM KCl, 1 mM MgSO4, 45 mM NaCl, 2 mM CaCl2

PBST: 10 ml 10X PBS, 2.5 ml 20% Triton X-100, 0.2 ml 0.5 M EDTA, pH 8, 87.3 ml ddH2O

DABCO: 50 ml total, 5 g DABCO, 5 ml PBS, 45 ml glycerin