Schematic of the total DNA staining experiment. (A) RPE-1 cells are incubated with IdU for 30 min followed by staining with anti-IdU (green) and anti-DNA antibody (anti-ssDNA, blue) to detect the integrity of the DNA flanking the DNA replication tract. (B) Predicted outcomes for sister chromatid alignment (left) versus separation (right). If sister chromatids remain aligned/adjacent to each other (left), they will result in DNA fibers that are blue–green–blue if there is no breakage, breakage at a, or breakage at b, and in fibers that are blue–green upon breakage at both at a and b. In contrast, if the sister chromatids become separated (right), they will result in DNA fibers that are blue–green–blue if there is no breakage, blue–green–blue and blue–green upon breakage at a or b, and blue–green upon breakage at both a and b.