FIG. 4.

MVV RNA expression in afferent lymph cells. (A) Cytospins of CD14⁻ FACS-separated afferent lymph dendritic cells from MVV-infected sheep were probed for gag mRNA by in situ hybridization using a digoxigenin-labeled gag riboprobe. Positive controls were skin fibroblasts heavily infected in vitro with MVV; as negative controls, all samples were hybridized with a sense gag riboprobe (data not shown). (B to D) Cytospins of metrizamide gradient cells from MVV-infected sheep were immunostained for CD1b and probed for tat RNA by in situ hybridization using a digoxigenin-labeled riboprobe. MVV-infected monocyte-derived macrophages, used as controls, were immunostained for MHC class I and for tat RNA. Specificity was verified using an isotype control antibody for the immunostaining (data not shown); for the in situ reaction, all samples were hybridized with a sense tat riboprobe. Red staining indicates positive signal for CD1b or MHC class I, and black staining indicates a positive signal from the in situ hybridization reaction. (B) MVV-infected macrophages stained for MHC class I expression and hybridized with sense tat control riboprobe. (C) MVV-infected macrophages stained for MHC class I expression and hybridized with antisense tat riboprobe. (D) Metrizamide gradient afferent lymph cells stained for CD1b expression and hybridized with antisense tat riboprobe. Arrows indicate double-stained cells.