Shown is a chromosome spread of a PtK1 cell pulsed with BrdU in early S phase, then treated with colcemid to arrest cells at metaphase. Spreads were immuno-labeled with FITC-conjugated anti-BrdU antibodies and counterstained with propidium iodide (red). Replication is confined to discrete sites of replication, some of which appear to occur in bands (arrows).
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.
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