Fig. 9. Time course of Cx43 and GFAP expression in differentiating neurospheres. Floating neurospheres, expanded for 10 days with EGF, were transferred onto coated coverslips and allowed to differentiate for 30 minutes (A,B), 3 hours (C,D) and 8 days (E,F). Double immunostaining was performed using a rabbit polyclonal antibody against Cx43 (red) and a mouse monoclonal against GFAP (green). Pictures were taken from three different neurospheres and are representative of three independent experiments for each time point. Images in A, C and E result from the merging of 14 confocal planes, whereas single plane views of the same neurospheres are shown in B, D and F. At each adhesion time, Cx43 was expressed by cells located within the sphere. Cx43 expression increased with time in cells migrating out of the sphere. GFAP expression was detectable only in a few cells at 30 minutes of adhesion but was strongly enhanced in the outgrowing layers at 3 hours and 8 days of adhesion. Bar, 10 µm in A-B; 30 µm in C-F.

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