Expression pattern of cultured, rat derived neurospheres. (A)Neurospheres were collected on glass slides by cytospin centrifugation, fixed and stained for Nestin, Sox2, DCX, Musashi, L1, LeX, PSA-NCAM and A2B5. DNA was stained using SYTOX (green). Most of the cells analyzed were positive for Nestin, Sox2, DCX, Musashi, Notch-1 (data not shown) and Notch-2 (data not shown) and negative for L1, LeX and the differentiation marker A2B5 for oligodendrocyte progenitors. Moreover, we detected moderate expression of PSA-NCAM, a marker for migrating neuronal precursor cells. Bar, 50 μm. (B) Neurospheres express both TNF-receptors. Neurospheres were collected, fixed and stained for TNFR-I and TNFR-II. Nuclei were stained with SYTOX (green). Note the high expression of both TNFR-I and II. (C) RT-PCR confirms expression of both TNF receptors. M: Marker, lane 1: TNFR-I, lane 2: TNFR-II. Lanes 3 and 4 are negative PCR controls without template.