Immunocytochemistry for Stem Cell Characterization

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).