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Cells of passage 4 were cultured in the complete NSC proliferation medium, immunostained with nestin (a, pink) or SOX2 (b, pink), and then counterstained with Hoechst 33342 (blue) as the nuclear marker. More than 90% of the plated cells stained positive for both nestin and SOX2 at passage 4, demonstrating that they were NSCs.
