Mass-isolation of egg-chambers
- Flies were fed with fresh yeast and kept for 1-2 days at 25°C.
- Mixed sex flies were narcotized with CO2 for a maximum of 5 minutes before proceeding to step 3.
- Narcotized flies were immediately immersed in 4% Formaldehyde in PBS (for FISH experiments) or in PBS supplemented with 0.1% Tween-10 (for ovarian extract or total RNA isolation).
- Flies were rapidly processed twice through a grinding mill adaptor at a fine setting (grade step "3") on a standard food processor (Kitchen Aid).
- The ground flies were size-separated using 850, 450 and 212µm sieves successively, resulting in a flow-through highly enriched for separated egg-chambers of all stages.
- Collection of mass-isolated material:
- For FISH experiments the co-isolation of testis and gut materials did not disturb the subsequent analysis and the material was allowed to settle by gravity and to be fixed for additional 15 minutes in 4% Formaldehyde, resulting in an overall fixation time of 20 minutes. The supernatant was then removed, the material washed twice in 1xPBS and then transferred stepwise into 100% methanol for storage at -20°C.
- For isolation of total RNA we manually selected egg-chambers at early stages (germarium to stage 7, previtellogenesis), late stages (stage 9-10, postvitellogenesis) and full ovaries highly enriched for stage 11+ egg-chambers using a stereomicroscope. For each stage we collected at least 10µl of total material that was frozen immediately.
- For preparation of ovarian extract mass-isolated egg-chambers were collected in a 50ml reaction tube, and briefly spun down and excess supernatant removed.