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Fig. 4. Progression of the replication bands analyzed by the double pulse-chase experiment. (A) Outline of the procedure. Clone-22 cells were pulse-labeled for 10 minutes with IdU, chased in fresh medium for varying times, pulse-labeled for 10 minutes with CldU, and further chased with fresh medium. The cells were harvested 4 hours after the IdU pulse-labeling, and subjected to metaphase spread preparation. The sites of IdU and CldU incorporation were detected using specific antibodies, and are shown in red and green pseudo-color, respectively. The HSR was detected by FISH using a biotinylated plasmid probe and Alexa 647-conjugated streptoavidin, which is shown in blue pseudo-color in this figure. The chase times between the IdU and CldU pulse-labeling are noted in the CldU panels. (B) Summary of the frequencies of HSRs labeled with both IdU and CldU at the varying interval time; (C-I) representative images. The orientation of the chromosomes detected with DAPI-staining (not shown) are indicated with thin lines.
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