Nestin ab6142

Immunocytochemistry was performed on trypsinized NPCs grown in 50 µg/mL poly-D-lysine-coated and 10 µg/mL fibronectin-coated 24-well plates containing defined medium [(5:3 mixture of DMEM low glucose (Life Technologies): neurobasal medium (Life Technologies), 0.5 mM 2-mercaptoethanol, 2mM L-glutamine, 5 IU of penicillin, and 5 µg/ml streptomycin (life technologies) supplemented with 1% N2 supplement (Life Technologies) and 2% B27 supplement (Life Technologies) without growth factors. After 5 days in culture, cells were fixed and stained with primary antibodies (Nestin: ab6142 (Abcam), GFAP: G9269 (Sigma-Aldrich), Tuj1: MMS-435P (Covance), and O4: MAB345 (Millipore)) at 4°C overnight. For fluorescence detection, Alexa Fluor-tagged secondary antibodies (Molecular Probes) were used and cells were counterstained with DAPI. Images were acquired using Metamorph software on a Nikon Eclipse TE300 microscope equipped with an optical camera (Optronics). Investigators were blinded to mouse genotypes and percentages of positive cells were calculated using the total number of cells in each image (DAPI nuclear stain). Data were analyzed with Prism 5 software (GraphPad Software) using a two-way ANOVA test as all data met the common assumptions (normally distributed, equal variances, independent samples) required for this test.