22 × 22 mm No. 1.5 coverslip (Marienfeld) was placed in a ceramic rack, placed in a beaker with NaOH (3 M) and sonicated for 10 min. The rack was dipped-and-drained in a beaker with MilliQ water, transferred to clean MilliQ water, and sonicated for 10 min. Finally, the rack was transferred to a new beaker with clean MilliQ water and sonicated for another 10 min. Coverslips were spin-dried and stored in a clean rack and sealed container until final use.
For experiments shown inFigs. 1 , 2 , and 3 , ovaries were dissected in a drop of halocarbon oil (Voltalef 10S; Arkema) placed on a clean coverslip, using tweezers to separate individual germaria. For experiments shown in Figs. 4 , 5 , and 6 , ovaries were dissected in a drop of Schneider’s medium supplemented with 10% FBS and 200 μg/ml insulin. Dissected ovaries were incubated for 2 × 30 s in 20 μl of supplemented Schneider’s medium. Finally, ovaries were transferred to a drop of supplemented Schneider’s medium on a clean coverslip next to a drop of halocarbon oil, and individual germaria were pulled into the oil. This latter protocol improved the sample lifetime and allowed for longer time-lapse imaging.
For experiments shown in