Lsm510 meta confocal laser microscope

Floating coronal brain sections (25 μm) were washed in 0.1 M PBS for 30 min and then blocked with 10% normal donkey serum with 0.1% Triton X-100 in PBS at room temperature for 1 h, as previously described by our laboratory (Zhang et al., 2009 (link)). Sections were then incubated with the corresponding primary antibodies at 4°C overnight. The primary antibodies used in this study were the following: rat anti-BrdU (1:200), mouse anti-NeuN (1:500, EMD Millipore, Billerica, MA), goat anti-DCX (1:50, Santa Cruz Biotechnology), goat polyclonal anti-MAP2 (1:100, Santa Cruz Biotechnology), mouse anti-Ki67 (1:200, Developmental Studies Hybridoma Bank), mouse anti-GFAP (1:200, Proteintech) and rabbit anti-Iba1(1:200, Proteintech). Sections were washed 3 times and were subsequently incubated with secondary antibodies (Alexa Fluor 568 donkey anti-mouse or rabbit, and Alexa Fluor 488 nm donkey anti-mouse or rat) at room temperature for 1 h. Sections were then mounted with a water-based mounting medium with anti-fading vectashield mounting medium for fluorescence with 4, 6- diamidino-2-phenylindole (DAPI) (H-1200; Vector Laboratories, Inc., CA, USA) and were cover slipped afterwards. Images were captured using a LSM510 Meta confocal laser microscope (Carl Zeiss).