For Immunofluorescence staining, paraffin-embedded tissue sections were subjected to a heat-mediated antigen retrieval procedure. To be specific, after sections were dried, dewaxed, and hydrated, they were heated to repair antigens. Antigens repair was performed with citric acid buffer (PH6.0) in a 92 °C water bath for 60 min and then restored at room temperature. Following that, tissue sections were incubated overnight with a primary antibody [(Col-2 pAb, #28,459–1-AP, Proteintech, Wuhan, China), (Sox-9 Rabbit mAb, #82,630, Cell Signaling Technology, MA, USA)] at 4 °C. The secondary antibody was then added to the sections, which were incubated for 60 min. The nucleus was counterstained with 75% DAPI (C0060, Solarbio) for 10 min. Finally, anti-fluorescence quencher was applied to seal the sections.
Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and then blocked with 3% BSA (A8010, Solarbio). The primary antibodies were then incubated overnight in PBS at 4˚C, followed by an hour of incubation with the secondary antibodies at room temperature. The rest of the steps are the same as above.
Cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and then blocked with 3% BSA (A8010, Solarbio). The primary antibodies were then incubated overnight in PBS at 4˚C, followed by an hour of incubation with the secondary antibodies at room temperature. The rest of the steps are the same as above.