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-BrdU antibody and an appropriate fluorochrome labeled 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 1A in, Ma, H et al. 1998 Spatial and temporal dynamics of DNA replication sites in mammalian cells. J Cell Biol 143:1415-1425.