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Krause lab protocols

Cuticle preparations

This procedure was adapted from one described over the phone (thanks P.G.). We're not sure if it appears elsewhere in published form.

-Collect embryos on apple juice-agar plates for suitable time period from a well-stocked cylinder or cage. Allow to age for 24-36 hr at 25o.

-rinse unhatched larvae into a nytex screen. Dechorionate in 3% bleach until embryos float to the surface (1-5 min). Don't worry about over-dechorionating. Rinse with water.

-remove screen and dip into a scintillation vial containing 5ml PBS/5ml heptane. The embryos should slide off. Those with vitelline membranes intact will stay at the interphase, while hatched larvae will settle to the bottom. Use a paint brush to remove remaining larvae from the dechorionating vessel and dip into heptane layer to dislodge from brush.

-using a 1ml pipetteman, suck up vitelline containing larvae from interphase. Eject any of the lower aqueous phase sucked up in the process. Transfer embryos in upper heptane solution to an eppendorf tube. Adjust volume to approximately 0.5 ml and add an equal volume of methanol.

-Close cap and shake vigorously for 15 seconds to devitellenize. The majority of embryos will settle to the bottom. Remove most of upper phase without removing any embryos. Shake once again. Now all of the embryos should settle to the bottom. Remove liquid and wash 2-3x with methanol.

-larvae at the bottom of the scintillation vial are generally wild type. They can also be drawn up in the aqueous solution in a P-1000 and transferred to an eppendorf tube. Remove the majority of liquid and add around 0.5 ml of methanol. Larvae should settle. Rinse 2X more with methanol. Larvae can be pooled with those above if desired.

-again using a P-1000, transfer the larvae to a clean glass slide. Use the excess methanol to disperse the larvae evenly by adding drops over clustered larvae. Allow the methanol to air dry briefly and then add a drop of Hoyer's/lactate. Cover carefully with a suitably sized coverslip, taking care to avoid bubbles.

-place in a 65o oven overnight to clear the larvae. Once cleared, flatten the larvae as follows. Wrap in a layer of aluminum foil. Place on a flat surface, cover with a second glass slide and place a lead pig such as those which come with radioactive substances. With this weight, larvae should be suitably flattened within 1-4 hr. Take care not to move the coverslip while adding or removing the weight and foil, as this will destroy the cuticles. Upon removing the foil, excess Hoyer's should have been exuded from the slide. This can be cleaned away using first water and then ethanol squirted in a stream over the slide. This avoids physical contact which may move the coverslip. Allow the slide to air dry and seal the edges with your favorite nail polish. Once dry, the slide should be permanent and relatively resistant to physical abuse.

Hoyer's Mountant: Add 30 g of gum arabic to 50 ml distilled water, stir overnight. While stirring, add 200 g chloral hydrate in small quantities. Add 20 g glycerol. Centrifuge at least 3hr at 12000 g to clear. Add lactate to increase contrast and decrease clearing time. We find 1:4 works best.

Wild-type first instar larva cuticle preparation



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