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Immunofluorescence assays. For MDV immunofluorescence assays, CEFs were plated on poly-L-lysine coated 12 mm diameter glass dishes (12-well plates) at 8 ¡¤ 104 cells well/ml density in 10% FCS medium. The following day, medium was removed and MDV was inoculated at m.o.i. of 1 PFU/cell, in 2% FCS MEM, in the presence and absence of proteins. After 5 h of incubation at 37 C in 5%CO2, monolayers were washed twice with PBS and then fixed with 4% p-formaldehyde in PBS for 30 min at room temperature. After washing twice more, 0.1M glycine was added to neutralize eventual residual traces of fixative. Fixed cells were left overnight at 4 C in PBS. For intracellular detection of MDV-specific signals by fluorescence microscopy, cells were permeabilized with 0.1% Triton X-100 for 5 min to allow antibodies to enter the cytoplasm and interact with viral antigens. Fluorescein conjugated (FITC) anti-MDV chicken antibody was purchased from Eurobio (France). |
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Сµ¤Ä¾Ä¾(½ð±Ò+3, MolEPI+1): ºÜÈÈÐÄŶ 2012-02-28 16:32:46
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Сµ¤Ä¾Ä¾(½ð±Ò+3, MolEPI+1): ºÜÈÈÐÄŶ 2012-02-28 16:32:46
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