Metaphase-arrested PtK1 Cells were swollen, pre-fixed in methanol-acetic acid, dropped onto cover glasses, incubated with H2O to promote spreading, and air dried. After hardening by a 4 hr incubation at 65C samples were fixed with 70% ethanol at -20C. Spreads were denatured with 4N HCl for 30 min at room temperature, and processed for immunofluorescence using an anti-BrdU antibody and FITC-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 6 in, Ma, H et al. 1998 Spatial and temporal dynamics of DNA replication sites in mammalian cells. J Cell Biol 143:1415-1425.