Annexin V Staining

Annexin V has previously been reported to stain dying cells when injected into Drosophila pupae (44). Here, we describe a method we have developed to stain ovary tissue, modifying the cell culture protocol from Molecular Probes. We find that significant Annexin V labeling of nurse cells is first detected during stage 9, several hours before other apoptotic changes are apparent (Fig. 3).

1. Dissect ovaries into ovarioles as described in Subheading 3.1.1. Remove egg chambers from the muscle sheath surrounding each ovariole by gently sliding forceps back and forth along ovarioles and/or lightly squeezing out egg chambers (see Note 10).

2. Transfer the egg chambers to microcentrifuge tubes (see Notes 1 and 2) and keep on ice.

3. Remove DR and wash egg chambers with 500 ||L of 1X ABB.

4. Mix 200 |L of 1X ABB with 20 |L of Annexin V conjugate. Remove wash from egg chambers and add Annexin/ABB solution. Incubate for 15 min at RT while gently rotating.

5. Remove annexin/buffer and wash with 500 |L of 1X ABB.

6. Remove wash, add 100 |L fix and 600 ||L heptane and rotate for 10 min at RT.

7. Remove heptane/fix and wash twice with excess PBT.

8. Remove fix, add 100 ||L of Vectashield mounting medium with DAPI, and mount on slides.

9. Observe egg chambers under the fluorescein filter of a fluorescent microscope. Those undergoing apoptosis should display strong green staining by Annexin V on the surface. DAPI staining can help to identify stages of development by viewing UV filter.

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