Mouse anti sox2

Cells on tissue culture plates were fixed in 4% paraformaldehyde for 15 min, washed using phosphate-buffered saline (PBS) 3 times, and then blocked and permeabilized with 10% donkey serum and 0.3% Triton X-100 in PBS for 1 h at room temperature. Primary antibodies (in 1× PBS with 0.3% Triton X-100 and 10% donkey serum) were incubated overnight at 4 °C. The next day, the cells were washed three times with PBS and incubated with secondary antibodies (in 1× PBS with 0.3% Triton X-100 and 10% donkey serum) for 1–2 h at room temperature. Finally, cells were washed three times using PBS and stained with DAPI for 10 min at room temperature. The primary antibodies used in this study include mouse anti-OCT4 (Santa Cruz, Cat# SC-5279), mouse anti-SOX2 (BD Biosciences, Cat# 561469), rabbit anti-FOXA2 (HUABIO, China, Cat# ET1703-76), and goat anti-SOX17 (R&D, Cat# AF1924).

For flow cytometric analysis of EB-NPCs, cells were collected using Accutase and stained with mouse anti-Nestin, mouse anti-human Sox1, and mouse anti-Sox2 per manufacturer’s instructions using the Human Neural Lineage Analysis kit (BD). For immunofluorescence microscopy, EB-NPCs were seeded on Geltrex-coated slides, fixed with 4% paraformaldehyde, and stained with rat anti-Nestin (1:500; Millipore), rabbit anti-Sox2 (1:200; Epitomics), or rabbit anti-Pax 6 (1:50; BioLegend) before addition of respective secondary antibodies (goat anti-rabbit AlexaFluor 568 and goat anti-rat AlexaFluor 488; both from Thermo Fisher). For analysis of multipotency, EB-NPCs were cultured in neuronal differentiation medium (Neurobasal, 1X B27, 1X GlutaMAX), astrocyte differentiation medium (DMEM [Thermo Fisher], 1X N2, 1X GlutaMAX, 1% FBS [Atlanta Biologicals]), or oligodendrocyte differentiation medium (Neurobasal, 1X B27, GlutaMAX, 30 ng/ml T3 [Sigma]) for at least 14 days before being fixed with 4% paraformaldehyde and stained with mouse anti-beta-III-tubulin (1:500; Abcam), rabbit anti-GFAP (1:200; Thermo Fisher), rabbit anti-NG2 (1:200; Chemicon), or rabbit anti-Olig2 (1:100; Abcam). Slides were cover-slipped and mounted using VectaShield Hard Set Mounting Medium with DAPI (Vector Labs), and all images were captured using a Nikon Eclipse Ti inverted microscope.