Coverslip bottom dish

Whole ovaries were dissected from flies fed wet yeast paste for 3–4 d and maintained at 25°C. Ovaries were dissected in Grace's insect culture media supplemented with 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA) and penicillin/streptomycin (Life Technologies). Late-stage follicles (S10B–S13) were hand dissected and placed in a drop of media on a coverslip-bottom dish (MatTek, Ashland, MA). Live imaging of GFP-Fascin and mRFP-Nup107 was performed using Zen software on a Zeiss 700 LSM mounted on an Axio Observer.Z1 using an LD C-APO 40×/1.1 W/0 objective (Carl Zeiss Microscopy, Thornwood, NY). For FRAP experiments, GFP-Fascin was photobleached using 100% laser power of the 488- nm laser for 50 iterations. One hundred images were obtained in a time series (3 prebleach and 97 postbleach) with no delay between images. FRAP recovery curve analysis was performed using the FRAP Calculator Macro plug-in in ImageJ (Abramoff et al., 2004 ) on 18 individual nuclei from 15 follicles for whole nucleus bleach and on 8 individual nuclei from 8 follicles for partial nucleus bleach.

Whole ovaries were dissected from flies fed wet yeast paste for 4–6 d and maintained at 25°C. Ovaries were dissected in Stage 9 medium (Prasad et al., 2007 (link)): Schneider’s medium (Life Technologies), 0.6× penicillin/streptomycin (Life Technologies), 0.2 mg/ml insulin (Sigma-Aldrich, St. Louis, MO), and 15% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA). Ovarioles and individual early stage follicles (S3–9) were hand dissected and either placed in a drop of medium or embedded in 1.25% low-melt agarose (IBI Scientific, Peosta, IA) made with Stage 9 media on a coverslip-bottom dish (MatTek, Ashland, MA; Groen and Tootle, 2015 (link)). Live imaging of GFP-Actin was performed using Zen software on a Zeiss 700 LSM mounted on an Axio Observer.Z1 using a LD C-APO 40×/1.1 W/0 objective (Carl Zeiss Microscopy, Thornwood, NY). For FRAP experiments, GFP-Actin was photobleached using 100% laser power of the 488-nm laser for 50 iterations. From 100 to 200 images were obtained in a time series (three prebleach and the rest postbleach) with no delay between images. FRAP recovery curve analysis was performed using the FRAP Calculator Package plug-in in ImageJ (Abramoff et al., 2004 ). FRAP recovery curves of multiple nuclei were averaged and analyzed using Excel (Microsoft, Redmond, WA).