C. Elegans Embryo Staining¶
Contributed by Ian Chin-Sang, Queens University, ON, Canada
Staining protocol for C. Elegans embryos.
- Wash several plates of worms off with M9. Use microfuge tubes to spin worms. Wash 2X with 1ml M9 buffer. Pool worms into 1 or 2 microfuge tubes. Use the tubes that have siliconized to prevent loss of embryos.
- Add 1 ml of Bleach solution (1ml 10 N NaOH 8ml H20) to worm pellet. Vortex occasionally and let sit for 3min.
- Spin embryos at full speed for 30 sec. Aspirate off bleach solution and add 1ml of new bleach solution. Again vortex and let set for 3min. Try not to exceed greater than 10 min in the bleach solution as the embryos tend to get damaged.
- Wash 3X with M9 buffer (1ml each time). Be careful not to loose the embryos. They tend to stick to the tube. If they are sticking to the side of the tube try spinning at 6000 rpm for 2 min.
- On the final spin aspirate off all but 30ul of the M9 buffer.
- Add 200 ul 2X witches brew (with 2% BME). Mix.
- Add 70ul 10% paraformaldehyde solution. Mix.
- Freeze immediately in liquid Nitrogen for 1 min. At this stages embryos may be kept at –80C for several weeks until ready to use.
- Thaw on ice for for at least 20 min.
- Wash 1X with Tris Triton Buffer (2min)
- Wash 2X with Antibody Buffer A (10min each wash)
- Add primary antibody (usually 1:100- 1:500 dilutions)
- Let sit for 4 hours or overnight without rocking.
- Wash 4X with Antibody Buffer B (1ml each with 10min between each wash).
- Add 500 ul of Antibody Buffer A 0.5 to 1ul of secondary antibody (ie. FITC anti-rabbit)
- Let sit for at leas 2 hours at room temperature in the dark.
- Wash 3X with Antibody Buffer B.
- Resuspend embryos in 10% glycerol (25-50 ul) plus anti-fading reagent.
- Put 5-10ul on a slide to visualize under the microscope.
This method is based, with permission, on an original protocol available here.