Phalloidin staining of F-actin in embryos

  1. Grow up worms to gravid adult stage on high density or NGM plates. Wash off worms with M9. Add 10 ml M9 to the plates and use a rubber policeman to gently dislodge the eggs from the plates. Pipette eggs into 15 ml tubes. If you want earlier stage eggs, save adults and bleach, but this is not necessary for morphogenesis-stage embryos and the bleach can interfere with staining.
  2. Pellet eggs. Resuspend in ~10 ml M9 with 0.01% Tween 20. Remove supernatant. Repeat for 3 washes total. (The eggs are very sticky, so include tween or another mild detergent in the M9 to prevent them from sticking to the sides of the tube). Resuspend in 2 ml culture media.
  3. Pull up sample with 21g needle in 3 ml syringe. Push out with a 30 g needle. Repeat for total 3 X.
  4. Wash 1X with 1 ml 1X ES/Hepes (no tween). Transfer to a siliconized 1.5 ml eppendorf tube. (The siliconized tube coating helps prevent eggs from sticking; siliconized tubes can be purchased or made by dipping tubes in 2% dimethyldichlorosilane in 1,1,1-trichloroethane and drying).
  5. Pull off supernatant to 120 μl. Add a small amount of Chitinase. Incubate ~10 minutes. Watch eggs on coverslip. Tadpoles should hatch out of eggshells.
  6. Add 680 μl of FIX 1 directly to egg sample. Incubate 2 minutes. Spin for 30 s in small microfuge.
  7. Remove Fix 1 and add 1000 μl of FIX 2. Incubate 15 – 20 minutes. Wash 3X with 1 ml culture media. Let eggs settle after 3rd wash. Remove supernatant.
  8. Dry a 10 μl sample of Alexa-Phalloidin in the dark (molecular probes). Resuspend Alexa -Phalloidin in 300 μl of 1X PBS.
  9. Add 300 μl Alexa -Phalloidin in PBS to egg sample. Incubate 1 hour in dark.
  10. Wash 3 X with 1 ml culture media.
  11. Pull off supernatant to ~30 μl. Add 30 μl glycerol/anti-bleach. Mix. Spin.
  12. Place 4 μl of pellet/glycerol on slide. Add coverslip. Press very gently between paper towels to remove excess glycerol.
  13. Seal coverslip with nail polish.

(Vida Praitis, based on Costa et al., 1997)

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