Mouse igg1 anti neun

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.

At 1 week, rats implanted with microwires were transcardially perfused with PBS, 4% paraformaldehyde, and 20% sucrose. Following decapitation, the skulls were exposed and placed in 4% paraformaldehyde overnight at 4°C and then 30% sucrose overnight at 4°C. The brains were then extracted and stored in 30% sucrose at 4°C overnight or until the brains sunk to the bottom of the container. Brains were frozen at −20°C and cryosectioned transversely onto charged glass slides (VWR, PA, USA). Slides were thawed to room temperature and washed with PBS. The slides were incubated at room temperature in blocking solution (0.4% Triton-X, 4% goat serum in PBS) for 1 h. The following primary antibodies were used: rabbit anti-GFAP (1:1,000, DAKO, CA, USA), mouse IgG1 anti-NeuN (1:500, Millipore, CA, USA), and mouse anti-CD68 (1:500, Millipore, CA, USA). Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Slides were then washed in PBS and washing solution (0.4% Triton-X in PBS). The appropriate secondary antibody was applied for 1 h at room temperature, followed by DAPI for 15 min. Slides were washed again in PBS and washing solution, dried, and coverslipped with Fluoromount-G (Southern Biotech, AL, USA). Stained slides were imaged at 10× on a Zeiss Axiovert 200 M (Carl Zeiss, NY, USA).