Analyzing Neurogenesis and Neuronal Differentiation

Brain sections and staining experiments were performed as in19 (link) except those performed on Gsh2-Cre; RCE; Zeb2^fl/fl mice brains processed as in48 (link). Primary antibodies used are: Calretinin (rabbit, Swant, 1/1000), GFP (chicken, Aves, 1/500), mouse IgG1 anti-NeuN (Millipore, 1:100), rat Igg2a anti-BrdU (AbD Serotec (Oxford B), 1/1000). Images were taken using a fluorescence microscope (Axiolmager Z1, ApoTome system, Zeiss) except for Gsh2-Cre; RCE; Zeb2^fl/fl sections (Leica DMR microscope) and for spine density measurement (laser confocal scanning microscope, LSM510, Zeiss - magnification: 63x). Data in graphics are presented as mean ± s.e.m of values obtained on n samples (*P < 0,05. **P < 0,01, ***P < 0,001). For BrdU incorporation analysis, animals at 2 dpe were injected once with a BrdU solution (50 μg/g body weight, Sigma, Saint-Louis MO) 2 hours before perfusion. BrdU staining was performed after 15 min incubation at 37° in 2N HCl-0.5%. In Fig. 4c, Zeb2 expression level per transfected cell was assessed as follows. Transfected cells in the RMS were identified based on GFP expression. Quantification of Zeb2 staining was performed using ImageJ software on a single z-plan focused on the nucleus (chosen using DAPI staining). A ROI was subsequently drawn inside the nucleus area and the mean intensity of Zeb2 staining signal was then measured across the ROI.