Preparation of Cuticles Using Mass Devitellinization of Embryos.
1. Make a 0-6 hour embryo collection and age for 36 hours at 250C. Place fresh yeast paste in the center of the plate to attract hatched larvae and then remove with a spatula. The remaining unhatched embryos can be stored at 40C for several days before proceeding.
NOTE: For wild type cuticles, collect embryos for 1-2 hours, age for 18 hours and then harvest for cuticle preparation before larvae hatch.
2. Remove unhatched embryos from the periphery of the plate using a fine paint brush which has been cut to create stiffer bristles. Place the embryos in a small basket and wash thoroughly with distilled water to remove yeast paste.
3. Dechorionate embryos in 50% Clorox bleach for 3-5 minutes. Wash well with 0.1% Triton X-100 and then with distilled water.
4. Using the paint brush, transfer embryos from the basket to an Eppendorf tube containing 1 ml 100% MeOH. Wash embryos 2x with 100% MeOH.
5. Devitellinize embryos in 0.5 ml 100% MeOH + 0.5 ml heptane by gently but thoroughly inverting tube for 1 minute. (DO NOT SHAKE.) Allow devitellinized embryos to sink to the bottom of the tube (this will happen fairly rapidly). Using a Pasteur pipet, remove the interface and all floating debris.
6. Wash embryos 2x or more in 1 ml 100% MeOH by inverting the tube several times, allowing the embryos to settle and removing all floating debris. This step should be repeated until the MeOH is clear.
7. Transfer embryos to a basket by resuspending in 1 ml 100% MeOH using a blue tip. Blot the basket dry and place in a small petri dish containing a 3:1 solution of acetic acid: glycerol. Incubate at 60oC for 1 hour.
8. Allow the embryos to cool to room temperature in the dish of fixative. Then remove the basket and blot the excess acetic acid: glycerol solution with a Kimwipe.
9. Place 25 ml Hoyer's mountant in the center of a microscope slide. Transfer a small number of cuticles from the basket into the mountant using the stiff-bristled paint brush. Place a 22 mm square no. 1 coverslip over the drop of Hoyer's and allow to completely settle. Capillary action will draw the mountant to the edge of the coverslip. The minimum amount of Hoyer's should be used so that none is squeezed out at the edges.
10. Transfer the slide to a 60oC oven and gently place a 10 g weight in the center of the coverslip. Incubate at 600C for 6-8 hours and then replace with a 200 g weight. Incubate for 1-2 days at 600C. The additional weight produces flatter preps but should not be added too soon in order to avoid popping the cuticles.
11. View the cuticles under dark field to see the overall cuticle pattern and to observe gross changes (e.g. missing segments). Use phase contrast to see subtle details (e.g. ventral pits, Keilin's sense organs). Photograph using Tech Pan black and white film (TP135-36) at ASA 50 and/or Ektachrome 160 tungsten color slide film (EPT135-36) at ASA 160.
Reagents:
Hoyer's Mountant: Add 30 g Gum arabic to 50 ml distilled water.
Stir overnight or until completely dissolved. (cover with plastic wrap to prevent evaporation)
With continuous stirring, add 200 g chloral hydrate slowly in small quantities to avoid clumping.
Add 20 g Glycerol.
Centrifuge until mountant is clear and devoid of debris (3 hrs to overnight at 10000 RPM in a Sorvall SS-34 rotor).
Store at room temperature in tightly capped tubes sealed with Parafilm.
This recipe makes approx. 200 ml.