Click it edu imaging kit

The Click-iT EdU Imaging Kit (Invitrogen) was used to assay cell proliferation following the manufacturer's protocol. Briefly, Huh-7, Bel-7402, SNU387, and SNU449 cells were incubated with doxorubicin at its IC₅₀ with or without remodelin for 24 h, then 10 μM EdU was added 2 h before fixation, permeabilization, and EdU staining. Nuclei were stained with 5 μg/mL Hoechst 33342 (Invitrogen) for 30 min. Cell proliferation was assessed using the Click-iT EdU Imaging Kit (Invitrogen) according to the manufacturer's instructions.

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.

Four-day-old seedlings were transferred from 1/2 MS supplemented with 1% agar (w/v) and 1% sucrose (w/v) medium to split-agar hydrostimulating medium with 800 mM D-sorbitol at bottom right side and 10 μM EdU in both sides or to normal 1/2 MS medium with different concentrations of zeatin containing 10 μM EdU. For controls, seedlings were transferred to split-agar medium plates without D-sorbitol or normal 1/2 MS medium without zeatin but with 10 μM EdU in both sides. After 60 min incubation, seedlings were washed in 1/2 MS liquid medium (3 × 5 min) supplemented with 1% sucrose (w/v) to remove excessive EdU. EdU detection was performed after washing the seedlings in phosphate-buffer saline (PBS, pH 7.4) containing 0.5% (v/v) triton X-100 (2 × 5 min). The EdU detection cocktail was made according to the protocol from Click-iT^® EdU imaging Kit (Invitrogen). The fluorochrome used was Alexa Fluor^® 488. Samples were submerged into the cocktail for 30 min in the dark, followed by washing in PBS (pH 7.4, 3 × 5 min). Samples were then covered by PBS (pH 7.4) containing 8 µg/ml Hoechst 33342 (component G of Click-iT^® EdU imaging Kit, Invitrogen) and incubated for 30 min in the dark, followed by washing in PBS (pH 7.4, 3 × 20 min). The root tips ware imaged using a confocal laser scanning microscope (Leica TCS SP8). The fluorescence signals were measured by the imaging software.