In vivo analysis of glial cell phenotypes during a viral demyelinating disease in mice

For thin layer chromatography, lipid extracts and standards were loaded on Baker thin layer chromatography silica-gel plates and run in a chloroform/methanol/0.2% CaCI2 (60:40:9) solvent system. The plates were air dried and subjected to routine iodine or orcinol staining or to 04 immunostaining. For the immunostaining, plates were blocked for 1 h at room temperature with 10% FBS and 3 % BSA in MEM Hepes medium and incubated overnight at 4°C with 04 culture supernatant diluted 1:4 in MEM Hepes with 10% FBS. At~er washing in cold PBS, the plates were incubated 90 min with biotinylated goat anti-mouse IgG (H + L specific; dilution, 11200; Cappel Laboratories, Malvern, PAL washed, incubated with streptavidinperoxidase complex (Amersham Corp., dilution 1/200), and treated with di-aminobenzidin¢ and hydrogen peroxide. Control plates followed the same procedure except that 04 antibody was omitted. The silica gel plates used remained intact throughout the prolonged immersion in aqueous solutions and no special treatment for stabilization was required.