Histological Evaluation of Cartilage and Chondrocyte Analysis

After fixing in 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and embedding in paraffin, 5 μm sagittal sections of cartilage specimens were cut and stained with hematoxylin and eosin (H&E), safranin O solution, and toluidine blue (Sigma-Aldrich) for histological examination. After washing with PBS (Servicebio, Wuhan, China), chondrocytes grown on slides were fixed with 4% paraformaldehyde for 30 min. Cells were then cocultured with a fluorescence in situ hybridization probe targeting tRF-5009A. After washing with PBS, cells were stained with DAPI. Images were captured using a confocal microscope (LSM780; Carl Zeiss, Germany). Immunofluorescence was performed 48 h after transfection, as previously reported [30 (link)]. The primary antibody used was against mTOR (1 : 100; Cell Signaling Technology, Danvers, MA, United States), while the conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, United States) was a goat anti-rabbit IgG. Images were obtained using a confocal laser microscope (LSM 780, Zeiss) at different magnifications.