Embryos were placed back

Embryos were placed back Palbociclib supplier into the abdominal cavity, and mice were sutured and placed on a heating plate until recovery. Retroviral infections were performed using

a preparation of Moloney murine leukemia retrovirus expressing GFP under control of the CAG promoter (Jessberger et al., 2007). Lateral ventricles of E13.5 embryos were injected following the same protocol as for in utero electroporation. In utero infection of cortical progenitors was performed with a limiting dilution of GFP retrovirus in WT and KO mice at E13.5 (Jagasia et al., 2009). In these conditions, we consistently labeled a few isolated neurons and a couple of small groups of neurons, clearly isolated from each other (average number of cells per clones = 4.7; average number of clones per animal analyzed = 3.4; not found in the same section for instance or separated by at least 300 μm in depth). We focused our analysis on the isolated groups of radially oriented neurons in the somatosensory cortex that consisted of clonally related cells (Figure S4). Embryonic brains were electroporated at E14.5,

Protease Inhibitor Library and 300 μm embryonic brain slices were prepared at E15.5 using a Leica VT1000S vibrosector. Slices were cultured on confocal inserts (Millipore; 5 mm height) with 1.2 ml of Hank’s balanced salt solution (HBSS) supplemented with Eagle’s basal medium Oxymatrine and 5% of horse serum (Invitrogen). Time lapse confocal microscopy was performed using an LD Plan Neofluar 20×/0,40 with a Zeiss LSM 510 inverted

microscope by imaging multiple z stacks at preselected positions on a given set of electroporated slices. Repetitive acquisitions were performed every 20 min for up to 20 hr, and movies were assembled with Zeiss Zen imaging software. Thirty neurons per slice were randomly selected within the SVZ/IZ and tracked using the Manual Tracking plugin of ImageJ software. The position of each individual neuron was manually marked as the center of the neuron and recorded as XY coordinate on each image of 1,024 × 1,024 pixels. The length of the migration between consecutive time frames was calculated by converting the length of the XY vector formed between to time frames in microns (1.024 pixels = 300 μm). Following ephrin-B1 overexpression, a neuron was considered as “clustered” if it belongs to a group of at least five cells in contact with each other and “single” if its position is more than 10 μm away from a cluster at the end of the time lapse acquisition. The parameters of migration were calculated using homemade calculation software and Microsoft Excel 2011. Embryos were fixed by transcardiac perfusion with 4% paraformaldehyde (Invitrogen). Brains were dissected, and 100 μm sections were prepared using a Leica VT1000S vibrosector. Slices were transferred into PBS/0.

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