Anti sox1

Assays for immunofluorescence staining were performed as described previously [35 (link),48 (link)]. The following antibody was used in the immunofluorescence assay: anti-SOX1 (R&D Systems). Finally, DAPI was used for nuclei staining, and images were visualized using a fluorescence microscope (Leica, Wetzlar, Germany).

For immunohistochemical analysis, cells were fixed with 4% paraformaldehyde (PFA) in 120mM phosphate buffer (PBS), pH 7.4, permeabilized with 0.05% Triton X-100 in PBS, blocked with 10% goat serum in PBS and subjected to immunohistochemistry staining with primary and secondary antibodies diluted in the blocking solution.
For immunolabelling the following antibodies at indicated dilutions were used: anti-Nestin (1:400; BD Bioscience), anti-Sox1 (1:100; R&D Systems), anti-Sox2 (1:200; Abcam), anti-Pax6 (1:200; DSHB), anti-Ki67 (1:200; Vector Labs), anti-TuJ1 (1:400; Covance), anti-Map2 (1:200; Millipore), anti-DCX (1:200; Millipore), anti-GFAP (1:200; Millipore), anti-O4 (1:50, Sigma-Aldrich), anti-GABA (1:200, Abcam), anti-vGlut1 (1:200; Millipore), anti-TH (1:100, Millipore), anti-Synaptophysin (1:100; Millipore), anti-PSD95 (1:200; Invitrogen), anti-vimentin (1:5000; Abcam), anti-S100β (1:1000; Sigma-Aldrich), anti-aquaporin 4 (AQP4, 1:100; Santa Cruz Biotechnology) and anti-excitatory amino acid transporter 2 (EAAT2, 1:100; Santa Cruz Biotechnology), secondary Alexafluorophore-conjugated antibodies (1:1000, Invitrogen). DNA was stained using Hoechst 33258 (1:10000, Invitrogen). All cells expressing a particular marker were counted manually and normalized to the total number of cells.

Human iPSCs were differentiated into haematopoietic cells as described previously^{17 (link)}. Neural cell differentiation was performed in accordance with previous protocols^{60 (link)} with slight modifications. iPSC colonies were mechanically dissociated into small aggregate suspensions and then plated onto Costar six-well ultra-low-attachment plates (Corning, NY, USA) in DFK medium (hES medium with 10 μM SB431542, 2 μM Compound C and 1% penicillin/streptomycin) with 10 μM ROCK inhibitor (Y27632; Reagents Direct). Medium without ROCK inhibitor was changed on day 2, day 4, and day 6. On day 8, EBs were collected and plated on Matrigel-coated 12-well plates in DFN2D medium containing DMEM/F12, 50% Neurobasal medium, 0.5% N2 supplement, 0.5% B27 supplement, 1% L-alanyl-L-glutamine, 1% MEM nonessential amino acid solution, 1% glutamax-1,2-mercaptethanol, 1% penicillin/streptomycin and 20 ng/µl basic fibroblast growth factor. Medium was changed every 2 days for 6 days. On day 18, cells were dissociated using TryPLE Express (Life Technologies) and plated on 24-well plates. Passage was performed every 2 days. Immunohistochemistry was performed using the following primary antibodies: anti-Sox1 (R&D Systems) and anti-Nestin (eBiosciences).

Assays for Western blot, cell viability by MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, Inner Salt), anchorage-independent growth, and invasion were performed as described previously [47 (link),48 (link)]. Detailed descriptions are accessible in the Supplementary Materials and Methods. The following antibodies were used in Western blot: anti-SOX1 (R&D Systems, Minneapolis, MN, USA), anti-HES1 (Cell Signal, Danvers, MA, USA).

Immunocytochemistry was performed as described previously with some modifications^{18 (link)}. Cells were fixed with 4% paraformaldehyde/PBS and permeabilized with 0.5% Triton X-100/PBS for 15 min. Then, cells were blocked with 5% foetal bovine serum/PBS for 30 min. For primary antibodies, cells were incubated at 4 °C for 16 h with the respective antibodies, such as anti-SOX1 (1:100, R&D Systems), anti-SOX2 (1:100, R&D Systems), anti-PAX6 (1:100, Stemgent, San Diego, CA, USA), anti-Nestin (1:200, eBioscience, San Diego, CA, USA), anti-TUJ1 (1:1,500, Covance, Berkeley, CA, USA), anti-MAP2 (1:1,500, Millipore), anti-TBR1 (1:200, Abcam, Cambridge, UK), anti-GFAP (1:1,500, DakoCytomation, Carpinteria, CA, USA), anti-p62 (1:100, Abcam), anti-NeuN (1:100, Millipore), anti-cleaved caspase 3 (1:800, Cell Signaling Technology, Beverly, MA, USA), anti-GRP78/BIP (Abcam), or anti-GADD153/CHOP (Santa Cruz Biotechnology). Cells were washed with PBS and then incubated for 60 min with secondary antibodies such as Alexa Fluor 488, 555, 594, or 647 (Thermo Fisher Scientific). Nuclei were counterstained with Hoechst 33342 (Dojindo, Kumamoto, Japan). The cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific).