After fertilization, one cell-stage embryos were electroporated using 20 to 100 μg of each expression construct as described in Corbo et al, 199710 (link).
The embryo were rise at 16, 18 or 21°C in ASW; fixed at the desired stage following the protocol described in Wagner and Levine 201254 (link). The embryo were washed several times with 0.05% BSA in PBS before being mount using FluorSave™ Reagent (Millipore). Images were acquired with a Zeiss 880 confocal microscope with or without the Airyscan module, and a wide field Zeiss Axio Observer Z1/7 combined to the Apotome 2.0 module.
All electroporation were performed in duplicate or triplicate (see related figure legends). Between 18 and 610 embryos were recovered per condition. No specific randomization strategy was performed except the assignment of the fertilized eggs to the different conditions.
Live imaging was performed on a home-build two-photon microscope system. Embryos were anesthetized with 16mg/ml MS-222 in ASW (Sigma-Aldrich). They were placed in microwells casted in 1% agarose in ASW59 (link) and the imaging was performed at 18 degree from LTBII to LTBIII stage. The images were assembled using Fiji60 (link) and the final rendering obtained with Imaris (Bitplane, Switzerland).
The embryo were rise at 16, 18 or 21°C in ASW; fixed at the desired stage following the protocol described in Wagner and Levine 201254 (link). The embryo were washed several times with 0.05% BSA in PBS before being mount using FluorSave™ Reagent (Millipore). Images were acquired with a Zeiss 880 confocal microscope with or without the Airyscan module, and a wide field Zeiss Axio Observer Z1/7 combined to the Apotome 2.0 module.
All electroporation were performed in duplicate or triplicate (see related figure legends). Between 18 and 610 embryos were recovered per condition. No specific randomization strategy was performed except the assignment of the fertilized eggs to the different conditions.
Live imaging was performed on a home-build two-photon microscope system. Embryos were anesthetized with 16mg/ml MS-222 in ASW (Sigma-Aldrich). They were placed in microwells casted in 1% agarose in ASW59 (link) and the imaging was performed at 18 degree from LTBII to LTBIII stage. The images were assembled using Fiji60 (link) and the final rendering obtained with Imaris (Bitplane, Switzerland).
