Ki67 antibody

Cells were fixed with 4% paraformaldehyde (cat. no. AS1018; ASPEN Biotechnology) for 15 min, and then incubated with 0.1% Triton X-100 (cat. no. ST797; Beyotime) for 5 min. Later on, cells were blocked with 1% BSA (cat. no. 10735078001; Roche) for 30 min, followed by incubation with the Ki67 antibody (cat. no. ab92742; Abcam Cambridge, MA, USA) overnight at 4°C. After that, the cells were incubated with a secondary antibody (cat. no. ab150077; Abcam) at 37°C for 1 h. Nuclei were counterstained with DAPI (100 μl; cat. no. C1005, Beyotime, Shanghai, China). Subsequently, fluorescence signals were imaged with a fluorescence microscope (Carl Zeiss, Jena, Germany).

Automated immunohistochemical
staining was performed using VENTANA BenchMark XT (Roche, Basel, Switzerland)
with UltraView Universal DAB Detection Kit (0526980600, Roche) and K_i-67 antibody (790-4286, Roche).

For immunohistochemistry (IHC), 8 μm sections of formalin-fixed and paraffin embedded brain tissues were first de-waxed and rehydrated before antigen retrieval. The SOX9-antibody (1:100 dilution; Abcam, Cambridge, USA) and Ki67-antibody (1:100; Roche, Basel, Switzerland) were used for this study. After incubation with the primary antibodies, the cells were rinsed and incubated for one hour with Biotin-labeled secondary antibodies at room temperature (Molecular Probes 1:800). Nuclei were stained by Hematoxylin. Stained sections were examined under a light microscope and the positive cells in five high power fields (1×200) were counted for statistic study.