Dopaminergic differentiation of SVZ-derived progenitors on PLL- and nECM-coated coverslips. (A) SVZ-derived neurospheres were seeded both on PLL- and nECM-coated coverslips and differentiated in the presence of a dopaminergic medium (including NGFβ, BDNF, Shh and FGF8b) for 10 or 35 days, respectively. Double stainings were performed with anti-MAP2 (red) and anti-TH (green) antibodies and nuclei counterstained with Hoechst (not shown). Colocalization of both markers is shown in the Merged (large) panels. All images are representative of three independent experiments. Scale bar, 40 μm. (B-C) Z stack analysis of cells differentiated for 10 days in PLL (B) or nECM (C). Upper panels (grey) show cell profiles, demonstrating cell incorporation into the matrix; lower panels (blue) show stack as a volume with Z value as 10 μm in PLL or 50 μm in nECM (see also Additional file 1 Video S1 and Additional file 2 Video S2). (D-F) To estimate the percentage of MAP2 and TH positive cells in each substrate and time condition, MAP2 (D) and TH (E) positive cells were determined. To obtain the ratio of TH positive neurons (F), TH positive cell number was divided by the MAP2 positive cell number. At least six different fields out of three independent experiments were quantified in each medium, substrate and time point. Statistical analyses were done by using two-way ANOVA assay followed by Bonferroni’s posttests (*P < 0.05; ** P < 0.01; *** P < 0.001).
García-Parra et al. BMC Neuroscience 2013 14:48 doi:10.1186/1471-2202-14-48