Cells were plated on poly-ornithine/laminin coated coverslips and grown for two days. BrdU (Sigma, B5002) was added to a final concentration of 10 µM for 2 hours at 37°C. Cells were fixed in 2% paraformaldehyde for 15 minutes at room temperature, and then permeablized in 95% methanol for 20 min at −20°C. DNA was denatured with 2 N HCl at room temperature for 10 minutes, neutralized in 0.1 M borate buffer (pH 8.5) for 10 minutes. Cells were blocked in 5% goat serum/0.2% Triton X-100/1% BSA/PBS at room temperature for 30–60 minutes. Cells were stained with mouse anti-BrdU (1:100, Sigma, B8434) and rabbit anti-NESTIN (1:2000, R&D Systems, MAB1259) antibodies. Cells were then stained with secondary antibodies goat anti-mouse Alexa 488 and goat anti-rabbit Alexa 555 (Molecular Probes Invitrogen, A21428). Coverslips were mounted with Vectashield and imaged. In 3 independent experiments, 300–500 cells were counted and percent BrdU incorporation was calculated as the total BrdU positive cells divided by the total NESTIN positive cells in a field. Statistical significance was calculated using ANOVA. CHIR-99021 effects on proliferation were measured in parallel
