Alkaline phosphatase staining (Supplementary Fig. S1a ) was performed using the Leukocyte Alkaline Phosphatase kit (Sigma).
The cultures of iPSCs and MNs were immunostained following standard procedures39 (link). Briefly, cells were fixed in 4% paraformaldehyde for 15 min at 4 °C, washed with PBS, and incubated in a blocking buffer (10% donkey serum and 0.2% Triton X-100 in PBS) for 60 min at room temperature before being incubated in primary antibodies (Supplementary Table S2 ) overnight at 4 °C. Appropriate fluorescently conjugated secondary antibodies were used to reveal the binding of primary antibodies (1:1000, Jackson, West Grove, PA) and nuclei were stained with DAPI. Images were collected with a Nikon TE600 fluorescence microscope (Nikon Instruments, Melville, NY) or a Nikon C1 laser-scanning confocal microscope (Nikon, Tokyo, Japan).
To quantify the population of OLIG2+, MNX+, and ChAT+ cells among total cells (DAPI labeled) or neurons (TuJ1+), images were imported into ImageJ (NIH) for analysis. Cell counting was performed by a person blind to the experiment and replicated in different cell lines in three independent experiments.
The cultures of iPSCs and MNs were immunostained following standard procedures39 (link). Briefly, cells were fixed in 4% paraformaldehyde for 15 min at 4 °C, washed with PBS, and incubated in a blocking buffer (10% donkey serum and 0.2% Triton X-100 in PBS) for 60 min at room temperature before being incubated in primary antibodies (
To quantify the population of OLIG2+, MNX+, and ChAT+ cells among total cells (DAPI labeled) or neurons (TuJ1+), images were imported into ImageJ (NIH) for analysis. Cell counting was performed by a person blind to the experiment and replicated in different cell lines in three independent experiments.
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