Based on our previous experiment (Liu et al., 2016 (link)), the mice were perfused transcardially with 0.9% saline followed by 4% paraformaldehyde (PFA) after deep anesthesia with pentobarbital. The brain was collected and dehydrated completely in 30% sucrose solution with 4% paraformaldehyde at 4°C. Brains were sliced coronally (30 μm thick) and stored at -20°C in cryoprotectant solution. Immunostaining was performed according to our previous method. In short, the brain sections were incubated with a rabbit anti-doublecortin (DCX) (1:1000, Cell Signaling) primary antibody in 1% bovine serum albumin (BSA) (12 h, 4°C). For BrdU staining, the sections were pretreated with 2 N HCl for 30 min at 37°C and then incubated with mouse anti-BrdU (1:500, BD Biosciences). After that, the sections were incubated with biotin-conjugated secondary antibody (1:200, goat anti-rabbit; Invitrogen) or Cy3 (1:500; donkey anti-mouse; Jackson ImmunoResearch) (2 h, 37°C), then treated with avidin-biotin peroxidase complex (Dako) for immunohistochemistry or 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime, China) for immunofluorescence. The BrdU- or DCX-labeled cells were observed and photographed with a Zeiss (Oberkochen, Germany) Axivert microscope equipped with a Zeiss AxioCam digital color camera connected to the Zeiss AxioVision 3.0 system.
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