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

Before immunolabeling, the sections were treated as follows: 10 rain in 100% ethanol at -20°C; three rinses in PBS at room temperature (1-2 min each); 5 rain in 1% sodium borohydride in PBS; and PBS rinses until bubbles were no longer observed (,o5-10 rain). Both ethanol and sodium horohydride treatments improved staining for CNP antigen and 04 antibody. In addition, sodium borohydride treatment served to eliminate an otherwise substantial tissue autofluorescence. The sections were then incubated for 3 h at room temperature in a solution containing 0.5 M Tris buffer, pH 7.6 (TB), 10% BSA, 3% normal goat serum, 0.1% gelatin, and 0.05% sodium azide to reduce nonspecific antibody binding. They were then exposed overnight to the primary antibody solution at 4°C. The primaries, applied singly or in combination, were 04, a mouse monoclonal IgM (dilution 1/2 of culture supernatant, from hybridoma kindly provided by I. Sommer; Sommer and Schachner, 1981) ; rabbit anti-CNP (dilution 1/100 of whole serum kindly provided by E A. McMorris et al., 1984; Sprinkle et al., 1980) ; and a monoclonal rat anti-GFAP (an lgG; dilution 1110 of culture supernataut from hybridoma 2.2B10.6 kindly provided by V. Lee et al., 1984) .