Ex vivo electroporation and live imaging were performed as previously described 29 (link), 30 (link). Ex vivo electroporation was performed on injected mouse embryos’ heads similarly to in vivo electroporation. The electrical parameters were the following: 50V, 50 msec length, 5 pulses, 1 sec interval. Following electroporation, brains were dissected in L15 (PAA Laboratories) and transferred into liquid 3% low melting agarose (Sigma) and incubated on ice. Embedded brains were cut coronally (300 μm) with a vibratome (Leica), and slices were transferred onto sterilized culture plate inserts (0.4-μm pore size; Millicell-CM, Millipore) and cultivated in complete Neurobasal containing Neurobasal medium (Gibco) supplemented with 1% B27, 1% N2, 1% glutamine, 1% penicillin/streptomycin and 1% fungizone.
Thirty hours after ex vivo electroporation, GFP was imaged in live brain slices using 900nm multiphoton excitation (Spectraphysics MaiTai DeepSee) with a Leica SP5 confocal scanner on a DM6000 CFS upright microscope. A 10x,0.4NA (dry) objective was used and reflected excitation collected with a non descanned PMT through a 525/50 filter (Semrock).
Movies were analysed using the Fiji software 34 (link). Three dimensional sample drift over time was corrected with the Correct 3D drift plug-in and cell movement was analysed using the Manual Tracking plug-in.
Thirty hours after ex vivo electroporation, GFP was imaged in live brain slices using 900nm multiphoton excitation (Spectraphysics MaiTai DeepSee) with a Leica SP5 confocal scanner on a DM6000 CFS upright microscope. A 10x,0.4NA (dry) objective was used and reflected excitation collected with a non descanned PMT through a 525/50 filter (Semrock).
Movies were analysed using the Fiji software 34 (link). Three dimensional sample drift over time was corrected with the Correct 3D drift plug-in and cell movement was analysed using the Manual Tracking plug-in.
