Shown are 0.5 micrometer thick confocal image of midplane sections of synchronized 3T3 fibroblasts pulsed for 2 min with CldU and labeled with FITC (green) followed by a variable chase time, and a second pulse with IdU and subsequently labeled with Cy-5 (red). With simultaneous pulses, the green and red signals overlap (A), but with longer chases, the two sets of replication sites become increasingly separate (B-F).
Cells were fixed with 70% ethanol at -20C, DNA denatured with 4N HCl for 30 min at room temperature, and processed for immunofluorescence using anti-CldU antibody and an appropriate FITC labeled secondary antibody. IuD was detected with a Cy-5 conjugated secondary antibody. Cells were examined in a MRC-1024 confocal microscope mounted on a Nikon Optphot 2 microscope with 60x 1.4 NA objective lens. See: Fig 3 in, Ma, H et al. 1998 Spatial and temporal dynamics of DNA replication sites in mammalian cells. J Cell Biol 143:1415-1425.
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