Immunocytochemistry

NSPCs plated on poly-D-lysine and laminin-coated glass coverslips were immunostained following established protocols^6,20. Briefly, after fixation in 4% PFA, cells were washed and blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich) in 1× PBS (Life Technologies) containing 0.4% Triton-X-100 (Sigma-Aldrich) and 2% normal goat serum (Life Technologies). After overnight incubation at 4°C with primary antibodies, cells were treated with appropriate secondary antibodies (1:500) coupled to fluorochromes Alexa 488, 594, or 647 (Life Technologies-Molecular Probes, Grand Island, NY, USA) and counterstained with DAPI. Primary or secondary antibodies were deleted under control conditions. The concentration of the primary antibodies used were as follows: nestin (1:300, EMD Millipore, Billerica, MA, USA); neuronal class III beta-tubulin (Tuj1, 1:300: Covance, Princeton, NJ, USA); GFAP, 1:500 (EMD Millipore); RIP (1:500, EMD Millipore); Nrf2-H300 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA); glutamate–cysteine ligase modifier subunit (GCLM; 1:200, Santa Cruz Biotechnology); and BrdU (1:100, Abcam, Cambridge, MA, USA).