Computational protocol: Asymmetric parental genome engineering by Cas9 during mouse meiotic exit

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Protocol publication

[…] Immunocytochemistry, differential interference contrast microscopy (DIC) and fluorescence imaging were essentially as described previously. In brief, mII oocyte and 1-cell embryo samples for the images in were incubated overnight at 4°C with mouse anti-α-tubulin (1:2000 [v/v]; Sigma) or -H1 (1:1000; Santa Cruz, USA) antibodies, followed by a 1 h incubation at 37°C with secondary antibody (1:250; Life Technologies Ltd., UK) conjugated to Alexa 488. DNA was stained by incubating samples at 37°C for 20 min in propidium iodide (1:200; Sigma, USA). Fluorescence was visualized on an Eclipse E600 (Nikon, Japan) microscope equipped with a Radiance 2100 laser scanning confocal system (BioRad, USA). Images were processed with ImageJ ( analysis software. Embryos were imaged on an Olympus IX71 equipped with an Andro Zyla sCMOS camera and OptoLED illumination system (Cairn Research Ltd., UK) and processed using Metamorph software (Molecular Devices, LLC, USA). Excitation at 484 nm with an ET-EYFP filter system was used to detect eGFP epifluorescence. Imaging of blastocysts for and was following culture in drops of KSOM (Specialty Media, USA) under mineral oil in 6 cm dishes in a humidified 5% CO2 (v/v air) incubator at 37°C. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens