1. Drosophila oogenesis is dependent on environment, age, and mating status of the female. Therefore, for both BrdU labeling and nuclear flow sorting, it is critical to use mated females of optimal age that are well fed and watered. This entails conditioning females on wet baker's yeast (the consistency of creamy peanut butter) for 2 d, followed by one more day on fresh wet yeast (a total of 3 d of conditioning). The females should have eclosed from the pupal case at least 3 d and no more than 8 d before the day of dissection (4-6 d post eclosion is optimal in most cases). Because oogenesis proceeds apace only if females have recently mated, males should be present during conditioning.

2. Treat eppendorf tubes and Pasteur pipets with 5% BSA solution to discourage sticking of the ovaries. Pipet 1 mL of 5% BSA into a tube, close the cap and invert, remove the BSA to the next tube to be treated, and so on. Treat pipets by pipetting the solution up and down once.

3. DNase I denaturation is preferred if ovaries are to be labeled for BrdU and an antibody to a protein because most epitopes are not detected after HCl treatment. After formaldehyde fixation in Subheading 3.1., step 6, substitute the following for steps 7-9. Wash twice, 15 min each, in PBS + 0.6% Trition-X; wash twice, 15 min each, in DNase buffer (66 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 1 mM of 2-mercaptoethanol added fresh), incubate ovaries in 100 |L of DNase I

(12.5 units/mL DNase buffer) at 37°C for 30 min (DNase I: Roche cat. no. 776785). Proceed to Subheading 3.1., step 10.

4. After collagenase treatment, the EBR may become slightly viscous, making it difficult for the ovaries to sink to the bottom of the Eppendorf tube. If this occurs, gently centrifuge the ovaries in a microfuge for 2-3 s to bring them to the bottom of the tube.

5. Crude but effective filters can be easily generated by supergluing a small section of Nitex mesh onto the end of a 1.5-cm section from a 5-mL plastic pipet that has been cut for this purpose. These sieves fit snuggly into 1.5-mL Eppendorf tubes.

6. As an alternative to DAPI, the nuclei can be stained with the nucleic acid dye propidium iodide. However, because propidium iodide (PI) stains both RNA and DNA, it is necessary to remove the RNA before analyzing the nuclei. After resus-pending the nuclei in 500 ||L of 1X nuclear isolation buffer in Subheading 3.2., step 10, add 5 |L of a 10-mg/mL RNase A solution to obtain a final concentration of 100 |g/mL. Incubate for 15 min at room temperature. Next add 2.5 |L of a 1-mg/mL PI stock solution to the nuclei. Let the nuclei stain for at least 15 min at room temperature before the analysis. The RNase A digestion and PI staining can be done concurrently. To obtain a flow cytometry profile, examine the propidium iodide stained nuclei using an argon 488 nm laser collected through a 545/642 bandpass filter.


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