Figure 5: Indirect and direct immunofluorescence staining focusing development and differentiation in S. aurata brain cultured cells. (a) GFAP immunoreactivity at the tips of the astrocytes processes after 3 week of culture (×200); (a’) Vimentin labeled fiber; Radial glia stained immunopositive for both glial fibrillary acidic protein (GFAP) and vimentin at 3 weeks old cultures; (a’’) DAPI counterstained nuclei corresponding to the same picture of (a) and (a’) (400x). The proliferative capacity of cells was shown by mitotic nuclei (white/cyan) double stained with Ab anti-PCNA-FITC and counterstained with DAPI (blue); the white dashed lines showed the actively dividing nuclei within the fiber extension. (a’’’) Phase contrast image showing basal RDG cells generating neurons (white dashed line showed the nuclear migration and fiber extension). (b-b’) Intense fluorescence for Nestin here appeared to be highly expressed in the processes by most IPCs, 3 weeks old cultures. (b’’) βIII-tubulin label immature neurons. (b’’’) NeuN was expressed nearly by mitotic figures. The mature post-mitotic neurons expressed in high density NeuN positive processes. (c-c’’’) Double immunofluorescence demonstrating colocalisation of neurons and astrocytes within the radial glial colony. (c) Several clusters of proliferative cells were observed (white and yellow circles); in particular, the central zone showed an intense granular production of neurons (UCHL1+/DAPI+/PCNA+) (white) inside the center of the colony of radial glia (GFAP+) (yellow circle). (c’) 3 weeks old cultures, highly immunostained with UCHL1 (green), showed newborn neurons migrate along neuronal fiber. (c’’) The outer boundary of the radial glia colony showed differentiated astrocytes with long GFAP positive filopodia (red) and neuronal fibers (c and c’), whereas in the inner zone there are clones of mitotic radial glia (c’’’, PCNA+/DAPI+). Scale bar = 50 μm.