Computational protocol: The RNA-binding protein ATX-2 regulates cytokinesis through PAR-5 and ZEN-4

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Protocol publication

[…] Embryo dissection was performed in Shelton’s Growth Medium, and embryos were mounted on 2% agar pads (made with egg salt buffer) in a drop of Shelton’s Growth Medium (). The pads were covered with a 22 mm × 22 mm coverslip and sealed in place with Vaseline. We found that this modified mount abrogates the osmotic defect observed in atx-2 fRNAi or atx-2(ne4297) embryos while allowing for optimal imaging during the first two cell divisions. Time-lapse images were taken every 10 s using a 200M inverted Axioscope microscope (Carl Zeiss, Oberkochen, Germany) equipped with a spinning-disk confocal scan head (QLC100; VisiTech, Sunderland, United Kingdom), an Orca 285 differential interference contrast (DIC) camera (Hamamatsu, Japan), and an Orca ER camera (Hamamatsu, Japan). The DIC and Orca ER cameras were operated through MetaMorph software (version; Molecular Devices, Sunnyvale, CA). Image rotating and cropping was performed using Fiji (ImageJ) software (). Fluorescence intensity of ZEN-4–GFP was obtained by drawing a region of interest of equal area around the spindle midzone of each embryo. These regions of interest were then used to measure the average pixel intensity. Background fluorescence obtained from a nonfluorescent region in the embryo anterior was subtracted. […]

Pipeline specifications

Software tools MetaMorph, ImageJ
Application Microscopic phenotype analysis