4.5. Fluorescent In Situ Hybridization (FISH)

FISH analysis was performed as described previously [62,80]. Briefly Mutu cells were treated with 0.075 M KCl for 20 min at 37 °C, fixed in methanol:acetic acid (3:1) for 30 min at room temperature and spread on the slides. Slides were treated with 4 × SSC (1 × SSC; 0.15 M NaCl, 0.015 M sodium citrate) containing 0.5% (v/v) nonidet P-40 for 30 min at 37 °C, dehydrated in a cold ethanol series (70, 80, 90%) for 2 min each, air dried and denatured in 70% formamide-2 × SSC for 2 min at 72 °C. Slides were dehydrated in a cold ethanol series and air dried. Hybridization probes for detection of EBV genomes were generated by nick translation with the plasmid containing EBV BamHI-WWYH fragment using biotin-11-dUTP (Roche, Basel, Switzerland). 20 µg of a probe was precipitated by ethanol in the presence of 6 µg salmon sperm DNA (Eppendorf, Hamburg, Germany) and 4 µg human Cot-1 DNA (Thermo Fisher Scientific, Waltham, MA, USA), resuspended in hybridization buffer (2 × SSC, 50% formamide, 10% dextran sulfate) and incubated for 10 min at 70 °C, for 5 min at 4 °C and for 1 h at 37 °C. A hybridization mix containing 5 ng probe was placed on each sample and incubated overnight at 37 °C in a moist chamber. Slides were washed in 2 × SSC containing 50% formamide for 30 min at 50 °C and in 2 × SSC for 30 min at 50 °C. After blocking in 4 × SSC containing 1% BSA, the hybridized probe was revealed by incubation with streptavidin conjugated to FITC (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C. Slides were washed twice in 4 × SSC containing 0.05% Triton X-100 for 5 min at room temperature. The nucleus was counterstained with Hoechst 33342 (Cell Signaling Technology). The copy numbers of EBV genome were analyzed by a confocal laser scanning microscope. (Fluoview FV10i, Olympus, Tokyo, Japan) and acquired by using FV10-ASW software Ver. 4.2 (Olympus, Tokyo, Japan).