Fig. 3. Nestin and UCH-L1 expression in undifferentiated and differentiating NPCs at 2, 6, 12, 24 and 48 hours. (A) NPCs were immunolabeled with antibodies against nestin and UCH-L1 in the proliferating phase (+bFGF; at 48 hours) or the differentiation phase (-bFGF; 2, 6, 24, 48 hours). Cultures were counterlabeled with Hoechst nuclear dye to facilitate cell quantification. (B) Quantitative analysis of the percentage of cells stained with each antibody. Nestin-positive cells gradually decrease as differentiation proceeds. The UCH-L1 expression level is both high (arrowheads) and low (arrows) in nestin-positive cells at 6 hours. Each experiment was analyzed by counting cells in three independent wells at the indicated times. The experiments were repeated at least two times. Bars, 50 µm.