Notes

1. Use no-stick microcentrifuge tubes (USA Scientific, Inc., Ocala, FL) to minimize the amount of tissue adhering to the side of the tube.

2. To move embryos or dissected tissue from plates to staining tubes and from staining tubes to slides, use plastic pipet tips from which 3/16-in. of the end has been removed with a razor. Rinse the tip in PBT just before using, hold the pipet vertically at all times and pipet very slowly. Glass Pasteur pipets may also be used, but tissue must only be held in the tip portion of the pipet, otherwise tissue may adhere to the inside of the pipet.

3. Cell death occurs from stage 11 until the end of embryogenesis. The pattern of cell death is dynamic and it is often useful to compare embryos that are approximately the same age. Embryos can be collected for a short period of time (0-3 h) and then aged to the desired stage. Embryos can be allowed to develop at 18°C overnight, which takes approximately twice as long as development at 25°C.

4. To maintain the integrity of imaginal discs and avoid tissue loss, it is helpful to leave all discs as one piece of tissue with any connections that remain following dissection. Individual discs can be separated upon completion of the procedure.

5. To change solutions in which embryos or tissues are incubating, use glass pipets drawn out to a fine tip. Remove most of the liquid from the samples and then carefully touch the tip to the meniscus, moving it slowly toward the sample.

6. Embryos may be transferred directly from the mesh to the fix with a fine paintbrush. Alternatively, embryos may be washed in the baskets with 0.1% Triton-X in water. The embryos are then pipetted to an empty tube and allowed to settle to the bottom of the tube. Remove the 0.1% Triton-X solution and replace with heptane/fix.

7. For acridine orange staining, it is critical that there is no trace of detergent (such as Triton-X) present when embryos are washed. Detergent will completely abolish AO staining.

8. It is essential that the tubes containing embryos be shaken very hard by hand. Standard rotation of the tubes is not sufficient for the heptane/AO to permeabilize the vitelline membrane.

9. Do not allow embryos or ovary tissues to dry out when the heptane is removed, as they will shrivel up quickly. Heptane evaporates rapidly; blowing gently on the slide will speed up its evaporation.

10. It is critical that the muscle sheath be removed from egg chambers because the Annexin V is not able to penetrate the unfixed muscle sheath. Furthermore, Annexin V stains the muscle sheath, which can affect the interpretation of results.

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