Co-replicative labeling of early-replicating RDs for correlative confocal-STORM imaging. (A) Schematics of the experimental approach. NRKs were harvested from a mitotic shake-off and plated in the presence of aphidicolin to arrest them at the G1/S transition. After 10 h, aphidicolin was removed and replaced by culture medium containing the fluorophore of interest (Fluorophore #1; in this paper, ATTO 633) coupled to dUTP. Cells were scraped off the culture chamber bottom, allowing the labeled dUTP to enter and be incorporated only in the actively co-replicating RDs (green circles); the rest remained unlabeled (gray circles). After labeling, cells underwent several rounds of division to allow a sparse localization of CTs within the nucleus (typically 3–4 d after the co-replicative labeling procedure). For dual-color experiments (bottom row), cells were subsequently scraped in the presence of a second fluorophore (Fluorophore #2; in this paper, ATTO 565) coupled with dUTP to label actively co-replicating RDs at a different time point (green and magenta circles). The interval Δt separating the application of Fluorophores #1 and #2 was varied between 0 and 120 min. (B and C) Examples of cells with an ATTO 633-dUTP single-color labeled CT (B) and with ATTO 633- and ATTO 565-dUTP double-color labeled CTs (C). Left: Image of the nucleus with transmitted light (gray) overlaid with the fluorescence channel of labeled RDs (ATTO 633 green, ATTO 565 magenta). Bars: 5 µm; (magnified portion) 1 µm.