Technical Reference #1847

1847.

I-BAR protein antagonism of endocytosis mediates directional sensing during guided cell migration Gabriel A. Quinones; Janet Jin; and Anthony E. Oro, Stanford University School of Medicine, Journal of Cell Biology, 189(1847), (2010)
Link To Paper

Materials & Methods:
Drosophila egg chamber live imaging For live confocal imaging egg chambers from ovaries were prepared according to previous protocols (Bianco et al. 2007; Prasad and Montell 2007; Tekotte et al. 2007) with some modification. Egg chambers were placed in Petri dishes with glass coverslip bottoms (MatTek) surrounded by halocarbon oil 700 (Sigma-Aldrich) and covered by the gas-permeable membrane from a Petriperm plate (Grenier Bio). Females were taken 1–3 d after eclosion and fed fresh yeast 1 d before dissection. Dissections were performed in Schneider’s Insect medium (Invitrogen) supplemented with 15% fetal bovine serum (Sigma-Aldrich) 0.20 mg/ml1 insulin (Sigma-Aldrich) 0.6x penicillin/streptomycin (Invitrogen) and 3 mM FM4-64 (Invitrogen). Ovaries were removed washed in dissection medium and individual ovarioles were removed from the muscle sheath using fine dissection forceps. Imaging was performed using a microscope (LSM-510 Meta; Carl Zeiss Inc.) with a 340 1.3 NA Plan NeoFluor oil immersion objective. Two channels were acquired simultaneously: GFP (488-nm laser and 500–550-nm band-pass filter) and FM4-64 (488-nm laser and 560-nm long-pass filter). 7 sections were taken 5 μm apart with 5 min between stacks. The three to four sections covering the migrating cluster were merged for each time point using the Volocity 4 imaging software package (PerkinElmer). Videos were made using Volocity 4 and exported as Quick-Time (.mov) files at 5 frames per second. Egg chambers were chosen in which border cells had clearly delaminated or were already migrating. For dye uptake assays egg chambers were incubated in 9 μM FM4-64 dye in imaging medium for exactly 30 min before the first image was taken. For Dynasore (Sigma-Aldrich) inhibition of dynamin in the egg chambers dissected egg chambers were incubated in imaging medium containing 80 μM Dynasore for 1 h before the addition of FM4-64.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
Drosophila MIM