F-ara-EdU Labeling and Detection Protocol

F-ara-EdU labeling and detection was performed as previously described with minor modifications^{57 (link)}. Seedlings grown in 1/2 MS were transferred to 1/2 MS media supplemented with 0.2 μM F-ara-EdU (Invitrogen Click-iT^® EdU Imaging Kit). Whole seedlings or excised roots were fixed in 3.7% (v/v) formaldehyde solution for 1 day, treated with permeabilization buffer [0.5% (v/v) Triton-X in 1× PBS] for 20 min, and washed twice in wash buffer (3% BSA in 1× PBS). Then, the samples were incubated in click-iT reaction mixture (Invitrogen Click-iT^® EdU Imaging Kit; 1× Click-iT^® EdU reaction buffer, CuSO₄, Alexa Fluor^® azide, and 1× Click-iT^® EdU buffer additive) for 30 min and washed twice in wash buffer, followed by a final wash in 1× PBS buffer. All procedures were performed at room temperature. The samples were mounted on glass slides using 30% glycerol and imaged immediately using a Zeiss LSM 7 DUO confocal laser scanning microscope (Carl Zeiss, Germany), with a 40× oil immersion objective. At least 15 seedlings from three independent experiments were analyzed.