Toluidine blue staining solution

The fixed proximal end of the tibia was decalcified with 10% EDTA (pH 7.4), embedded, and sliced. Subsequently, the section was de-paraffinized, rehydrated, and stained with tartrate-resistant acid phosphatase (TRAP) and toluidine blue staining solution (Sigma–Aldrich, Inc., MO, USA), respectively. Histopathological images were collected using a microscope equipped (Nikon Corporation, Tokyo, Japan). Undergoing the grayscale transform, the toluidine blue slides were used for the histological analysis as previously described (Vidal et al., 2012 (link)), i.e., total area (T.Ar), calcified bone area (B.Ar), bone perimeter (B.Pm) can be obtained directly from images, and then the trabecular bone area (Tb.Ar), number (Tb.N), and thickness (Tb.Th) were calculated as the following equations. $\text{Tb}.\text{Ar}(\%)=\mathrm{B}.\text{Ar}/\mathrm{T}.\text{Ar}\times 100,$ $\text{Tb}.\text{Th}\left(\mathrm{\mu }\mathrm{m}\right)=2\times \mathrm{B}.\text{Ar}/\mathrm{B}.\text{Pm},$ $\text{Tb}.\mathrm{N}(\text{No}./\text{mm})=(\mathrm{B}.\text{Ar}/\mathrm{T}.\text{Ar})/\text{Tb}.\text{Th}.$

After digestion and resuspension, the chondrocytes were planted on slides and paraformaldehyde (4%) was added for fixation for 30 min and then 1% toluidine blue staining solution (Sigma-Aldrich) was added and stained for 2 h, followed by three times wash with PBS. Finally, neutral resin was added for sealing and slides were observed and photographed under a pathological microscope (CX41; Olympus, Tokyo, Japan).
Similarly, in the propidium iodide (PI) staining experiment, chondrocytes on slides were fixed and cleaned, and then PI working solution (Beyotime Biotechnology, Shanghai, China) was added for incubation for 30 min at room temperature. After being washed again, the cells were photographed under a fluorescence microscope (DFC450C; Leica, Wetzlar, Germany).