Cell light apollo stain kit

EdU (5 μg/g per day for 7 days) was injected intraperitoneally into the mice during the final week before sacrifice. EdU staining was performed using the protocol provided by the Cell-Light™ Apollo Stain Kit (RIBOBIO, C10371–1, Guangzhou, China). Following deparaffination, the sections were rinsed with a glycine solution for 10 min, permeabilised in phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 10 min, washed with PBS (3 × 10 min), and incubated at room temperature in the presence of the Apollo reaction cocktail for 30 min. Next, the sections on the slides were permeabilised with 0.5% Triton X-100 in PBS for 10 min and then rinsed with methanol (2 × 5 min). Finally, the slides were incubated with Hoechst at room temperature for 30 min. Nuclear counterstaining was performed using DAPI, and samples were mounted with 50% glycerine. Images were obtained by using fluorescence microscopy. The EdU- and DAPI-positive cells were determined using ImageJ software, and the EdU labeling index (a ratio of the number of EdU-positive cells to the number of DAPI-positive cells) was calculated. The threshold of fluorescence intensity was set according to the range in which these cells of interest can be detected. Two fields of view for each slide were analysed, using two slides from each knee and eight knees from each group.