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

After washing the sections with TB, secondary antibodies diluted in TGBA were applied to the sections for 3 h at room temperature. For double and triple stains, appropriate secondary antibodies were applied simultaneously. 04 was visualized with rhedamine-conjugated goat anti-mouse IgM (/~m chain specific; Jackson Immunoreseareh Laboralories, Inc., Westgrove, PA; 58 t~g/rnl), the rabbit anti-CNP with flnoresccin-eonjugated donkey antirabbit Ig (Amersham Corp., Arlington Heights, IL; 1:15), and the rat anti-GFAP with biotinylated sheep anti-rat Ig (Amersham Corp.; 10 t~g/ml). After washing in TB, sections were treated with 25 t~g/ml of streptavidin conjugated to 7-amino-4-methyl-coumarin-3-acetic acid (Molecular Probes Inc., Junction City, OR; Khalfan et al., 1986; Appel ct al., 1987) in TGBA to mark the biotinylated secondary antibody, washed again, fixed 1-4 min in 4% formaldehyde in PBS, and coverslipped with a solution of .02 M Tris, pH 8.6, and 80% glycerol. Sections were examined by epifluorescence with a Zeiss Photoscope Ill equipped with three simultaneously mounted filter sets for rhodamine, ffuorescein, and coumarin fluorescence and photographed with Kodak T-Max-400 film, developed for ASA 1,600.