Fig. 4. Visualization of replication sites within permeabilized primary or transformed human cells after nuclear run-on replication. (A) Controlled permeabilization with digitonin produces cells with intact nuclei (impermeable to large molecules that lack nuclear localization signal (e.g. Texas Red IgG, right panel) and well preserved nuclear structure. Intact nuclei prepared from WI38 (B) or HeLa (C) cells were introduced into in vitro replication cocktail and nascent DNA at replication forks generated in vivo was labeled with biotin-dUTP. Nuclei were fixed with 70% ethanol and biotin-dUTP was detected with Texas Red-conjugated streptavidin. DNA was stained with 0.1 µg/ml 4',6-diamidino-2-phenylindole (DAPI).