To trace the migration of cells from their place of origin, we injected in whole embryos the lipophilic dye carboxyl-fluorescein di-acetate (CFDA, SE, 557 M.W., V12883, Invitrogen; 10 mM diluted in DMSO 472301, Sigma-Aldrich) which only fluoresces upon cell entry (García-Moreno et al., 2010 (link)). Injections were made in rat embryos at E11.5 and in mouse embryos at 9.5 in different areas of the ventricular surface of the diencephalon or midbrain, followed by incubation for 24 h in blood serum obtained from Wistar rats with a constant flow of a gas mix of 95% O2 and 5% CO2. After incubation, embryos were fixed with 3.5% PFA in PBS for 4 h and immunostaining was performed as described above.
To quantitate the migratory cells from various regions of the midbrain, ventricular injections were applied at the locations indicated in Figure2K followed by TH immunostaining and flat-mounting. For each injection, Z-stacks of confocal images were obtained from regions containing all CFDA labeled cells that migrated away from the labeling point among which TH+ and TH− cells were counted. Results were analyzed with the program JASP 0.8.6.0 and JASB 0.9.0.1 applying an ANOVA with post hoc Tukey test. The number of injections analyzed for each region indicated in Figure 2 were: DI , 2; 1 , 3; 2 , 4; 3 , 3; 4 , 3; and 5 , 3.
To quantitate the migratory cells from various regions of the midbrain, ventricular injections were applied at the locations indicated in Figure