Uv 780 meta confocal microscope

For fluorescence immunocytochemistry, NSPCs adhered to different precoated coverslips were fixed with 4% paraformaldehyde in 0.01 M phosphate-buffered saline (pH 7.4) for 2 hours at room temperature and blocked with 5% v/v fetal bovine serum or with 0.5% v/v Triton-X 100 (Sigma-Aldrich, ×100) in PBS. NSPCs were incubated with rabbit polyclonal to Ki67 (Abcam, 1:200), rabbit polyclonal to Doublecortin (Abcam, 1:200), rabbit polyclonal to glial fibrillary acidic protein (GFAP) (Proteintech Group, Inc, 1:100), rabbit polyclonal to Olig2 (Proteintech Group, Inc, 1:100), rabbit polyclonal to Sox2 (Abcam, 1:200), or mouse monoclonal to Nestin (Abcam, 1:250) overnight at 4 °C and then goat anti-rabbit Cy3 or anti-mouse FITC (Proteintech Group, Inc, 1:200), or donkey anti-rabbit FITC or anti-mouse Cy3 (Beyotime, 1:250) for 2 hours at room temperature. All cultures were counterstained with the DNA-binding dye 4′-6-Diamidino-2-phenylindole (DAPI, 2 mg/ml in PBS) for 10 minutes at room temperature. Then, Coverslips were mounted onto glass slides and the images were obtained from a Zeiss UV 780 Meta confocal microscope (Carl Zeiss), and examined by AxioVision 4 (Carl Zeiss AG) or Adobe Photoshop CS5 software.

Following the treatment, NSPCs or brain tissues were fixed using 4% paraformaldehyde at room temperature for 1h. After being blocked in 1% BSA, 5% goat serum and 0.2% Triton X-100 in PBS (PH 7.4) at 4°C for 2h, the specimens were incubated with with corresponding antibodies against against Ki67(1:1000, Abcam, UK), DCX (1:500, Abcam, UK), BrdU (1:500, Millipore, Germany), GFAP(1:500, Abcam, UK), Nestin (1:1000, Proteintech Group, Inc, US) at 4°C overnight. Then, cells were incubated with corresponding secondary antibody, goat anti-rabbit Alexa Fluor 488, goat anti-rabbit Alexa Fluor 555, goat anti-rabbit Alexa Fluor 647, Goat Anti-Rat Cy3 or anti-mouse Alexa Fluor 488 (1:200, Beyotime, China) at room temperature for 2h, followed by counterstaining with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI, 2 mg/ml in PBS) for 10 min. Finally, the images were captured using a Zeiss UV 780 Meta confocal microscope (Carl Zeiss) and examined by Adobe Photoshop CS6 software.
For BrdU immunostaining, brain sections were incubated in 2N HCl at 37°C for 30min. Stained sections were rinsed using 0.1M borate solution (pH=8.5) twice for 10min, incubated in 3% H₂O₂ for 30 min and blocked with 5% normal goat serum at room temperature for 1h.