Mouse knee samples were decalcified, dehydrated, and waxed before being cut into 5-μm-thick sections for immunofluorescence analysis. After dewaxing with xylene and gradient alcohol, sections were sealed with 10% goat serum at room temperature for 1 h and incubated overnight at 4 °C with different primary antibodies including anti-COL2A1 (1:200; Abcam; ab34712), anti-aggrecan (1:200; Proteintech; 13880-1-AP), and anti-ADAMTS5 (1:200; Abcam; ab182795). Each section was then rinsed there times with PBS, followed by incubation for 1 h with green or red fluorescence under ambient temperature. Sections were washed three times with PBS, and nuclei were stained with DAPI. Staining of each section was observed and recorded using fluorescence microscopy.
In immunofluorescence staining of cells, chondrocytes were inoculated in a 24-well plate. After chondrocytes of each group reached 70–80% confluence in the 24-well plates, they were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Chondrocytes were then blocked with 5% goat serum at room temperature for 15 min and incubated overnight at 4 °C with the following primary antibodies: anti-COL2A1 (1:200; Abcam; ab34712), anti-aggrecan (1:200; Proteintech; 13880-1-AP), and anti-ADAMTS5 (1:200; Abcam; ab182795). The same procedure was used for immunofluorescence staining of tissue.
In immunofluorescence staining of cells, chondrocytes were inoculated in a 24-well plate. After chondrocytes of each group reached 70–80% confluence in the 24-well plates, they were fixed with 4% paraformaldehyde for 15 min and washed three times with PBS. Chondrocytes were then blocked with 5% goat serum at room temperature for 15 min and incubated overnight at 4 °C with the following primary antibodies: anti-COL2A1 (1:200; Abcam; ab34712), anti-aggrecan (1:200; Proteintech; 13880-1-AP), and anti-ADAMTS5 (1:200; Abcam; ab182795). The same procedure was used for immunofluorescence staining of tissue.