EdU Incorporation Assay for Replicating Nuclei

EdU incorporation assay was performed as previously described [25 (link)]. The wild-type and gcn20–1 plants were grown on 1/2 MS agar plate for 5 days and then incubated in 1/2 MS liquid medium with 10 μM 5-Ethynyl-2′-deoxyuridine (EdU) (Invitrogen, Carlsbad, CA, USA) for 30 min. Then the seedlings were fixed for 30 min in 4% (w/v) formaldehyde solution in phosphate buffered saline (PBS) with 0.1% Triton X-100. Following 3 × 10 min PBS washes, the seedlings were incubated for 30 min at room temperature in EdU detection cocktail (RiboBio, Cell-Light™ Apollo stain Kit) followed by a 10 min rinse. Finally, the root tips of wild-type and gcn20–1 seedlings were imaged with Laser-scanning confocal microscope using the Argon laser 488 nm excitation and 478–553 nm emission lines. The fluorescent nuclei represent actively incorporating (replicating) nuclei.