Mouse anti human ki67

EBs were collected on day 12 of differentiation, washed with PBS, fixed with 4% paraformaldehyde for 20 min, then cryoprotected with 15% sucrose overnight before being embedded in OCT media. About 20~30 EBs were cryo-sectioned into 30 of 10 μm sections for immunofluorescent analyses. MGE tissues were first collected in Hibernate-E media on ice before processed just as EBs. For antibody staining, glass slides with sections were washed with PBS three times and permeabilized with 0.3% Triton X-100 before blocking with 2% skim milk (Difco). Primary antibodies were chicken anti-GFP (1:500, Aves Labs), mouse anti-Nkx2-1 (1:200, Leica microsystems), rabbit anti-Nkx2-1 (1:200, Santa Cruz Biotechnology, Inc.), and mouse anti-human Ki67 (1:200, BD Pharmingen). Alexa 488 and Alexa 594 secondary antibodies (1:500, Thermo Fisher) were used according to the primary antibody species. Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, 5ng/ml, Thermo Fisher).

NSPs were cultured on poly-L-ornithine coated coverslips and then fixed with paraformaldehyde (4% in phosphate buffer saline (PBS)), followed by a permeabilization with 0.3% Triton X-100 in PBS. After washing in PBS, the blocking step was done in PBS containing 3% BSA at room temperature for 30 min. The cells were then incubated overnight at 4 °C with primary antibodies, purified mouse anti-human KI67 (1:100; BD Biosciences, Allschwil, Switzerland #550609), rabbit anti-CD133 IgG (1:100; Abcam, Cambridge, UK #ab19898), rabbit anti-CD44 IgG (1:100; Abcam, Cambridge, UK #ab243894), and rabbit anti- NF-κB p65 (1:100; cell signaling technology, Danvers, MA, USA #8242). After washing, cells were incubated with the corresponding anti-rabbit or anti-mouse secondary antibodies conjugated to cyanine 2 or cyanine 3 for 1 h at room temperature (1:1000; Jackson ImmunoResearch, Cambridge, UK). For the GFAP staining, a cyanine 3-conjugated mouse anti-GFAP IgG antibody (1:800; Sigma C9205) was used. Cells were then washed and mounted with DAPI-Fluoromount G (SouthernBiotech, Birmingham, AL, USA). The immunoreactivity was assessed using a LSM 510 META inverted confocal microscope (Carl Zeiss Micro Imaging, Oberkochen, Germany) and analyzed on ImageJ software.