Cuticle Prep Protocol From Drosophila Protocols Textbook pg.

1. Allow flies to lay eggs on grape juice-agar plates in dark for 3 hours. Remove plate from flies and allow the eggs to develop at 25°C for a further 18 hours. 2. Transfer embryos to an egg sieve as follows: a. Place enough tap water in the petri dish to just cover the eggs. b. Brush over the surface of the grape-juice agar plate vigorously with a small paint-brush, breaking up any clumps of yeast. The eggs will soon be seen swirling in the water. Pour this water into the egg sieve. If eggs remain on the plate, add more water (repeat the brushing if necessary), and pour the water through the sieve. c. When all the eggs have been collected, wash them thoroughly under a stream of tap water. 3. Dechorionate embryos as follows: a. Place the egg sieve in a small dish containing 50% bleach for 2-3 minutes. b. Remove the egg sieve from the bleach; wash the eggs and container thoroughly with tap water. c. If sorting is required, sort at this stage and return sorted embryos to egg sieve. 4. Devitellinize embryos as follows: a. Dry the mesh of the egg sieve by blotting the bottom of the sieve on a paper towel. b. Dry the paintbrush on the paper towel and then swirl it around the bottom of the egg sieve. The embryos will stick to the paintbrush. c. Shake the brush in heptane, i.e., in the top phase of a 1:1mixture of heptane and methanol (500μl each) contained in a 1.5 ml microcentrifuge tube. The eggs will fall off the brush to the interphase. If large numbers of embryos have been collected, simply use a larger tube and volumes of heptane and methanol (1:1). d. Repeat steps 4b and 4c until all of the eggs are transferred. e. Close the microcentrifuge tube and, with extreme vigor, shake the tube for 30 seconds. This will cause the vitelline membrane to pop and the embryos will fall to the bottom of the tube. 5. Remove the heptane and methanol with a glass pipette. Replace with 1 ml of methanol. Repeat methanol wash twice. 6. Replace the final wash of methanol with 0.1% Triton X-100. Repeat once. 7. Mount cuticles as follows: a. Use a glass pipette to transfer the embryos with a small amount of liquid to a clean slide (previously cleaned with 70% EtOH). Fold a piece of filter paper (or a Kimwipe) to a point and use this to wick up most of the liquid, carefully avoiding the embryos. Try not to let the embryos dry out, although irreparable damage will not occur if they dry for a brief period.

. c. When most of the liquid is removed. but may overdigest the cuticles) d. Place the slide at 60°C for at least 1 hour. ( It may be preferable to let the slides cook for one to several days. Seal the edges of the coverslip with nail polish. as this hardens the Hoyer’s mountant.b. Place a coverslip onto the preparation. place a drop of Hoyer’s medium (or lactic acid:H2O [3:1]) onto the embryos. Spread out and arrange the embryos using a fine forceps.

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