In vitro analysis of the oligodendrocyte lineage in mice during demyelination and remyelination

Immunocytochemistry O-2A lineage cells were identified based upon three-color immnnofluorescence that enabled simultaneous visualization of reactivity with antigalactocerebroside (GC), anti-glial fibrlllary acidic protein (GFAP), and the 04 antibody. Anti-GC is a mouse monoclonal IgG3, kindly provided by B. Ranscht (La Jolia Cancer Research Foundation, La Jolla, CA), which recognizes GC and an earlier antigen emerging on the cell surface shortly after the appearance of 04 immunostaining (Ranscht et al., 1982; Bansal et al., 1989) . The rabbit polyclonal anti-GFAP, kindly provided by R. Pruss (Merrill Dow Pharmaceuticals, Cincinnati, OH), immunostains GFAP but does not react with other intermediate filament proteins . 04 is a mouse monoclonal IgM, kindly provided by 1. Sommer (Southern General Hospital, Glasgow, Scotland), (Sommer and Schachner, 1981) which recognizes sulfatide, seminolipid, and an unidentified antigen (Bansal et al., 1989) . 04 and anti-GC were supernatants from hybridoma cultures containing 10% FBS in DME. The supernntants were mixed together and diluted 1:4 and 1:2, respectively, in a MEM-Hepes buffer solution (0.1% gelatin, 1% BSA, and 0.05% sodium azide in MEM-Hepos) and applied to the fixed cells for 1-2 h. 04 was visualized with rhodamine conjugated goat antimouse IgM (25 t~g/mi; Jackson Immunoresearch Laboratories, West Grove, PA). Anti-GC was visualized with fluorescein conjugated goat anti-mouse IgG3 (16.6 tzg/ml; Fischer Biotech, Orangeburg, NY). These secondary antibodies were also mixed together in MEM-Hepes buffer solution and applied for 1-2 h. After washing, the cultures were fixed with 5 % glacial acetic acid in ethanol for 10 min at -20"C to expose internal cellular antigens. This was followed by three washes in 10% FBS MEM-Hepes and three washes in 0.5M Tris buffer. Polyclonal rabbit anti-GFAP was diluted 1:2,000 in a Tris buffer solution (0.1% gelatin, 1% BSA, and 0.05% sodium azide in 0.5M Tris buffer) for binding overnight at 4"C. Anti-GFAP was visualized with biotinylated donkey anti-rabbit IgG (10/~g/ml in the Tris buffer solution, 1-2 h; Amersham Chemical Co., Arlington Heights, IL) followed by streptavidin conjugated 7-amino-4-methyl-coumarin-3-acetic acid (25/zg/ ml in the Tris buffer solution, 1-2 h; Molecular Probes, Inc., Eugene, OR; Khallhn etal., 1986). Atter a final 5-rain fixation in 4% paraformaldeh)de the cultures were either coverslipped, with a solution of 20 % 0.02 M Tris buffer (pH 8.2) and 80% glycerol, or processed for autoradiography (see below).