Quantification of Differentiation Markers

The immunofluorescence staining and cell quantification were carried out as described previously [2 (link),27 (link)]. Briefly, the cell samples were washed with PBS for 30 min followed by blocking with PBS (3% normal goat serum, 0.1% triton X-100 in PBS) for 1 h at room temperature. Primary antibodies were applied overnight at 4 °C. The following primary antibodies were used: anti-rat BrdU (Cat# ab6326, Abcam), anti-mouse GFAP (Cat# Z0334, DAKO, Santa Clara, CA, USA), anti-mouse Tuj1 (Cat# G712A, Promega), anti-rabbit Caspase 3 (Cat# AB3623, Millipore, Boston, MA, USA) and anti-mouse MAP2 (Cat# M9942, Sigma). The second day, fluorophore-conjugated secondary antibodies were applied for 1 h at room temperature. Images were captured and the numbers of BrdU⁺, Tuj1⁺, GFAP⁺ and Caspase3⁺ cells were quantified using ImageJ software (NIH).