Samples were hybridized with the BamHI W fragment of EBV, a sequence approximately 3-kb long that is repeated 6 to 10 times in the EBV genome. After hybridization, the biotinylatedBamHI W probe was detected using the fluorescein-conjugated avidin-biotin amplification system (green signals). Human total genomic DNA was directly labeled with tetra-methyl-rhodamine-dUTP (red signal). The presence of yellow signals indicated overlap of the human and the EBV probes. Nuclei were counterstained with the DNA fluorochrome, DAPI (blue). Digital images were captured in gray scale and subsequently pseudo-colored using computer imaging software. (A) Namalwa (left) and BAE nuclei (right) hybridized simultaneously with the EBV BamHI W probe (green) and human genomic DNA probe (red). The Namalwa nucleus shows two distinct signals with the EBV probe and uniform hybridization with the human probe, whereas the bovine nucleus was negative for both probes. (B through E) These figures show four examples from the two-color FISH analysis of the presence of human genomic DNA (red) and EBV-DNA (green) in BAE cells cultured with irradiated Namalwa cells for 1 week. Yellow signals indicate overlap of the signals from the EBVBamHI W and the human probes. (F) Simultaneous analysis of presence of EBV-DNA and human DNA in a BAE nucleus after cultivation with the EBV-negative cell line BL41 shows the presence of human DNA but not of EBV DNA in the BAE nucleus. (G) BAE nucleus cultured with irradiated Namalwa cells showing uptake of human DNA (red). A positive signal was analyzed by digital confocal microscopy. Images were sampled in the z-axis and subsequently processed with 3-D rendering software (Openlab) to generate three-dimensional pictures. The nuclei and incoming DNA could be viewed at different angles (0°, 44°, 56°, and 72°) and depths that showed that the positive signal was localized within the nuclear cage.