Edu st067

About 1.6 × 10⁵ cells per well were inoculated in a 6-well plate, EDU (ST067, Beyotime) was added after 12 h of culture to make the final concentration 10uM and the cells were incubated for 2 h in an incubator. After removing the medium, 1ml fixative (4% Paraformaldehyde) was added, and cells were fixed at room temperature for 15 min. After removal of fixative, and 1ml permeabilization solution was added and cells were incubated at room temperature for 15 min. Later 0.5ml Click reaction solutions were added and cells were incubated for 30 min in the dark. Finally, cells were stained with Hoechst. Results of the EdU assay were observed and analyzed under an EVOS M5000 microscope (Thermo Fisher Scientific).

To evaluate angiogenesis, mice were injected (i.p.) with 5-ethynyl-2′-deoxyuridine (EdU, ST067, Beyotime, Shanghai, China) (50 mg/kg) on days 5, 6, and 7 following injury. EdU incorporation was then assessed using the BeyoClickTM EdU-488 kit (C0071S, Beyotime, Shanghai, China) on 5 µm coronal sections with immunofluorescence.