Immunostaining was performed according to the standard protocol.
For immunofluorescence analysis of the cartilage, frozen sections were incubated with antibodies specific for YAP (1:100, 14074; Cell Signaling), TAZ (1:100, 83669; Cell Signaling), MST1 (1:100, 14946; Cell Signaling), Collagen II (1:100, MA5-13026; ThermoFisher), and MMP13 (1:100, 18165-1-AP; proteintech) overnight at 4°C and then incubated with Cy3-conjugated secondary antibody (1:1000, BA1031, BA1032; BOSTER) for 1 h.
For immunofluorescence analysis of the primary chondrocytes, cells were fixed with 4% paraformaldehyde for 10 min, then incubated in 0.3% Triton X-100 for 15 min, blocked by blocking buffer (1% bovine serum albumin in PBS) for 15 min, and incubated with antibodies specific for YAP (1:100, 14074; Cell Signaling) and Collagen II (1:100, MA5-13026; ThermoFisher) and were then incubated with Cy3-conjugated secondary antibody (1:1000, BA1031, BA1032; BOSTER) for 1 h.
Images were captured with a laser-scanning confocal microscope (OLYMPUS FV1000) using the FV10-ASW 4.2 Viewer (OLYMPUS) and analyzed using ImageJ software.
For immunofluorescence analysis of the cartilage, frozen sections were incubated with antibodies specific for YAP (1:100, 14074; Cell Signaling), TAZ (1:100, 83669; Cell Signaling), MST1 (1:100, 14946; Cell Signaling), Collagen II (1:100, MA5-13026; ThermoFisher), and MMP13 (1:100, 18165-1-AP; proteintech) overnight at 4°C and then incubated with Cy3-conjugated secondary antibody (1:1000, BA1031, BA1032; BOSTER) for 1 h.
For immunofluorescence analysis of the primary chondrocytes, cells were fixed with 4% paraformaldehyde for 10 min, then incubated in 0.3% Triton X-100 for 15 min, blocked by blocking buffer (1% bovine serum albumin in PBS) for 15 min, and incubated with antibodies specific for YAP (1:100, 14074; Cell Signaling) and Collagen II (1:100, MA5-13026; ThermoFisher) and were then incubated with Cy3-conjugated secondary antibody (1:1000, BA1031, BA1032; BOSTER) for 1 h.
Images were captured with a laser-scanning confocal microscope (OLYMPUS FV1000) using the FV10-ASW 4.2 Viewer (OLYMPUS) and analyzed using ImageJ software.