Figure 7.

Double pulse–double chase experiments. Replication sites in synchronized mouse 3T3 fibroblasts were first labeled for 2 min with CldU (FITC secondary antibody, green sites), chased for 2 h, and then pulsed again for 5 min with IdU (Texas red secondary antibody, red sites). The cells were then chased a second time for (A) 7 h (late S- or G₂-phase); (B) 24 h (2nd cell generation); (C) 48 h (3rd cell generation); or (D–F) 12 h (M-phase). (D) Replication sites (green for early replication sites and pseudocolor red for later replication sites); (E) Chromosomes were counterstained with PI, and pseudocolored to blue. The secondary antibody for the IdU labeling was Cy-5 conjugated and pseudocolored to red. (F) Merged image of replication sites and chromosomes. One chromosome is enlarged 4× and indicated by the white boxed window. White and yellow arrows indicate replication sites forming chromosome band structures. Bars, 5 μm.