Computational protocol: Fat2 acts through the WAVE regulatory complex to drive collective cell migration during tissue rotation

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Protocol publication

[…] Light microscopic images of eggs were taken at an Axioplan 2 (Carl Zeiss) using a Plan-Neofluar 10×/0.30 objective. Fluorescently labeled specimens were analyzed on an AxioExaminer.Z1 microscope equipped with an LSM 710 confocal unit (Carl Zeiss) using a Plan-Apochromat 63×/1.4 oil-immersion objective. Further image processing was performed using the Zen 2009 software (Carl Zeiss). SIM images were taken with a CellObserver SD microscope equipped with ELYRA S.1 system using a 63×/1.4 oil-immersion objective (Carl Zeiss); five grid rotations were used with a mean of two as previously described. 4D datasets were analyzed using Imaris software (Bitplane).Live imaging of egg chambers was performed in Schneiders medium (Sigma-Aldrich) containing 0.2 mg/ml insulin (Invitrogen) and 15% FCS (Thermo Fisher Scientific) at room temperature as previously described (), using an inverted Observer.Z1 microscope equipped with a Yokogawa CSU-X1 spinning disk scanning unit (YOKOGAWA), an Axiocam MRm CCD camera (6.45 µm × 6.45 µm; Carl Zeiss AG) and a 488-nm laser line (LASOS). Egg chambers were imaged through a Plan-Apochromat 25×/0.8 (UV) Vis-IR oil-immersion objective, Plan-Apochromat 63×/1.4 oil-immersion objective or Plan-Neofluar 100×/1.30 oil-immersion objective (Carl Zeiss). ZEN software 2012 (Carl Zeiss) and Fiji (ImageJ) software was used for acquisition and processing of the images. Volume-rendered 4D reconstructions (gamma 1.0) were performed on the z-series (, E1–E4, and corresponding Video 7, A and B, were reconstructed based on 125 single sections over 15 min) was performed using Imaris software (Bitplane). […]

Pipeline specifications

Software tools Imaris, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Chemicals Tyrosine