Click itr edu kit

The 5-ethynyl-2′-deoxyuridine (EdU) assay was performed using the Click-iTR EdU Kit (RiboBio, China) according to the manufacturer’s instructions. Specifically, cells were plated in 96-well plates and treated after adhesion. A total of 100 μL of culture medium containing 50 mM EdU was added to each well, and, 4 h later, the cells were fixed with 4% formaldehyde for 30 min. After washing, the cells were incubated with a solution in the kit for 30 min and stained with Hoechst (RiboBio, China) to identify nuclei, and images were captured with a fluorescence microscope (Thermo Fisher Scientific, USA). EdU-positive cells were determined using ImageJ 1.50i software (https://imagej.en.softonic.com/).

A total of 100 µL of EdU culture medium (1:1000, 50 µM) was added to each well of the villous explants for 12 h, followed by fixing with 4 % formaldehyde for 30 min, paraffin embedding, and sectioning at 4 μm. After intensive washing, the tissue sections were subjected to a Click-iTR EdU Kit (RiboBio, Guangzhou, China) for 30 min and stained with 100 µL of DAPI (RiboBio) at room temperature for 30 min. Image analysis was performed on a fluorescence microscope (EVOS FL Color Imaging System). The relative percentage of EdU-positive cells was determined by examining three to five samples in three wells.