Live imaging experiments were performed as described in [41 (link)] with the modification of incubating dissected ovarioles in 2μg/mL Hoechst 33342 DNA dye (Invitrogen) for 10 minutes, and then rinsing them gently in dissection media. Images were acquired every 5 minutes for up to 6 hours using a Zeiss AxioImager Z1.1 and AxioVision software. For live imaging, we utilized lines that express membrane-tethered Green Fluorescent Protein specifically in border cells from a transgene. Specifically, we used stocks bearing either the slbo-Gal4, UAS-mCD8-GFP [41 (link)] or Slbo-lifeAct:GFP [17 (link)], which have been shown to have normal cell migration and ovary development. Stocks were maintained at 18 degrees Celsius under standard culture conditions [42 ]. Flies were fed extra yeast in vials at 29 C overnight, then moved to room temperature for several hours before being dissected. Flies used for imaging egg chambers with differing number of border cells were obtained from Bloomington Stock Center as described in [43 (link)].
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