Fig. 5. Replication sites of the HSR subchromosomal domain in the late S phase nucleus. Clone-22 cells were pulse-labeled with BrdU for 30 minutes, and then the cells were immediately fixed with PFA. The site of BrdU incorporation was detected with an anti-BrdU antibody in red (A'-C'); HSR was detected by hybridization with a DIG-labeled plasmid probe in green; merged images in A-C. In whole nuclear images (left image of each panel), approximate nuclear rim was outlined based on the faint background of BrdU-signal (light blue circles). The images show that these nuclei were in late S phase, because the BrdU labeling was restricted to the internal large heterochromatin islands. Serial confocal images of 0.68-µm intervals in the z-axis were obtained for each HSR domain (right panels). There were three types of BrdU-labeling: (A) at the peripheral region, (B) at the whole domain and (C) at the internal region. Bars, 5 µm.