Safranino fast green staining solution

The segments selected method for LM was the same as described above for SEM. The segments were first fixed using FAA (G1103, Servicebio, Wuhan, China), then dehydrated and infiltrated by paraffin. After that, process tissue samples in melted paraffin in cassettes and cut 4 µm sections using Leica RM2016 microtome (Leica, Shanghai, China). The sections were dyed by SafraninO-Fast Green Staining solution (G1031, Servicebio, Wuhan, China) after deparaffinizing and hydrate to water. At last, the sections were dehydrated and mounted with resin. Sections were cut to a thickness of 3 μm and then observed images with a NIKON ECLIPSE E100 microscope (NIKON, Tokyo, Japan). The cutinized cell walls were red color and the cellulose cell walls were green color. The epidermal cell size was observed with a NIKON DS-U3 (NIKON, Tokyo, Japan), at least five biological replicates for each sample. The experiment of was repeated for three biological samples from three differential stages.

Materials were taken from the right knee of mice, fixed with paraformaldehyde, tissue dehydrated, decalcified and paraffin embedded. Each cartilage sample was cut into thick sections of 5 µm. The sections were stained with safranin O‐fast green staining solution (Servicebio, Wuhan, China), images were acquired by using a Leica microscope with a LAS system, and the degree of OA was analyzed by using Mankin's score.