Secondary fl uorescein and rhodamineconjugated antibodies

To identify astroglia cell types, we used antibodies against GFAP (clone GF-01, 1:100, Exbio, Prague), and polyclonal sera to GFAP (1:100, Dako). Oligodendroglia cells were detected by antibodies to GalC (clone mGalC, 1:10, Boehringer-Mannheim, Vienna) and to O4 antigen (clone 81, 1:10, Boehringer-Mannheim, Vienna). Microglial cells were defi ned as immunoreactive with antibodies to CD11c (clone BU15, 1:50, Immunotech, France), and polyclonal sera to ferritin (1:50, Sigma). Neuronal cells were identifi ed with antibodies to MAP2 (clone HM-2, 1:50, Sigma), and NF (clone NF-01, 1:100, Exbio, Prague). The endothelial cells were detected by polyclonal sera against Von Willebrand Factor-VIII-related antigen (1:100, Dako). The antibodies to vimentin (clone V9, 1:100, Sigma), to cytokeratins monoclonal anti-pan CK (types: 1,4,5,6,8,10,13,18,19, 1:100, Sigma), and polyclonal sera against fi bronectin (1:100, Sigma) were used for further characterization of human brain cells. The secondary fl uorescein-and rhodamineconjugated antibodies were purchased from Sigma and Sevapharma (Prague).

To identify astroglial cells, we used antibodies against GFAP (clone GF-01, 1:100, Exbio, Prague). Neuronal cells were identifi ed with antibodies to MAP2 (clone HM-2, 1:50, Sigma), and NF (clone NF-01, 1:100, Exbio). The antibodies to vimentin (clone V9, 1:100, Sigma) and polyclonal sera against fi bronectin (1:100, Sigma) were used for further characterization of human "glia-like" cells. The secondary fl uorescein-and rhodamine-conjugated antibodies were purchased from Sigma and Sevapharma (Prague).
Cells grown on uncoated glass coverslips were used for indirect and double immunofl uorescence staining. The intermediate fi lament proteins (GFAP, vimentin, neurofi laments), MAP2 and fi bronectin were detected on cells fi xed in methanol-acetone (1:1) solution for 15 min. at (-5 °C). Cells for indirect immunofl uorescence were incubated for 1h with primary antibodies and 30 min with 1:50 diluted appropriate secondary antibodies. Double labeling was performed with primary, and afterwards with appropriate mixtures of secondary antibodies for 1 h and 30 min, respectively. Immunofl uorescence microscopy was performed using an Olympus BX51 microscope (Hamburg, Germany).