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Native BrdU staining

XW Xiao Wu
BW Bin Wang
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Cells were labeled with BrdU (10 μM) for 36 h53, washed with culture medium after labeling, and treated with CPT (1 μM, 1 h). Cells were then pre-extracted with pre-extraction buffer, fixed with 3% paraformaldehyde/2% sucrose solution, treated with cold methanol for 20 min at −20 °C, washed with PBS, and treated with cold acetone for 30 s. After blocking with 2% BSA for 1 h, staining was performed with BrdU antibody followed by Alexa-488-conjugated secondary antibody. Images were captured with 80i eclipse Nikon microscope using ×40 objective. BrdU nuclear intensity was measured by ImageJ software (ImageJ 2.0 and 64 bit Java8 from https://imagej.nih.gov).

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