Col x

After fixation with 4% paraformaldehyde, cells were blocked and permeabilized in 1% w/v bovine serum albumin (BSA, Sigma-Aldrich), 10% donkey serum (Life Technologies) and 0.1% Triton) for 30 min at RT. For nuclear antigens, cells were treated with 0.5% Triton (Sigma-Aldrich), then primary antibodies, CTNNB (mouse, 1:100; bdbiosciences), CK19 (rabbit, 1:500; abcam), HNF4a (rabbit, 1:100; abcam), AFP (mouse, 1:100; abcam) and ALB (goat, 1:100; Bethyl), ASGR1 (mouse, 1:100; Thermo Fisher Scientific), COL1 (rabbit, 1:500; abcam), Vimentin (rabbit, 1:200; abcam); CD44 (mouse, 1:200 abcam), CD31 (rabbit, 1:50; abcam); Col-X (mouse, 1:250; abcam), MRP2 (mouse, 1:200; abcam), ZO-1 (mouse, 1:100; abcam), CD26 (rabbit, 1:100; abcam), Pan-Cadherin (mouse, 1:100; Sigma), Claudin 1 (rabbit, 1:100; abcam), Occludin (mouse, 1:100; abcam), Stem121 (mouse, 1:100; Cellartis), phosphor-SMAD2/3 (rabbit, 1:100; Cell signalling technology) and GLI1 (rabbit, 1:100; abcam) were applied for overnight at 4 °C. After washes, cells were then incubated with Alexa 647, Alexa 568, Alexa 488 conjugated secondary antibodies or 488-Phalloidin (1:250; Life Technologies). Samples were counterstained with DAPI (NucBlue, Life Technologies). Confocal micrographs were captured using Nikon Ti spinning disk confocal microscope equipped with Andor Neo camera and images were processed by NIS-element software.

For immunofluorescence, frozen sections of chondrocyte spheres were washed with PBS for three times, these cell slices were permeabilized with cold 0.2% Triton X-100 (Sigma, USA) in PBS for 5 min. A step of enzymatic antigen retrieval with 0.1% Trypsin in PBS was performed prior to block for 1 h. After blocking, antibodies against Aggrecan (1:300; Abcam, UK), COL II (1:300; Abcam), SOX9 (1:200; Affinity), MMP13 (1:200; Affinity), COL X (1:500; Abcam), NANOG (1:300; Abcam), OCT4 (1:300; Abcam), TRA-1-60(1:300; Abcam) and Ki67 (1:300; Abcam) were added overnight. The following day, cells were washed three times with PBS and then incubated at 37 °C for 1 h with Alexa 594-conjugated goat anti-rabbit secondary antibody (1:300; Abcam) or Alexa 488-conjugated goat anti-mouse secondary antibody (1:300; Abcam). The cell nucleus was counterstained with DAPI (Beyotime, People’s Republic of China).

Paraffin sections of 4-week-old tibiae were rehydrated and digested in 0.1% trypsin for 10 min at the room temperature, and then treated with 3% H₂O₂ for 20 min. Sections were incubated with primary antibodies in PBS overnight at 4 °C. Col-X, MMP13, MMP9, cathepsin K, OPG, DKK1, and sclerostin antibodies were obtained from Abcam (Cambridge, MA, USA). Axin1 and β-catenin antibodies were obtained from Sigma (St. Louis, MO, USA). NFATc-1 and c-Fos antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Negative control sections were incubated with IgG (Beyotime Biotechnology, Shanghai, China). A Polink-2 plus polymer HRP detection kit (PV-9001, ZSGB-BIO, Shanghai, China) was used for incubation with secondary antibody and horseradish peroxidase (HRP)-streptavidin.

For immunohistological (IHC) staining, murine articular sections were deparaffinization and hydration, followed treated with parenzyme (0.25%) for 30 min. An additional 10-min treatment with H₂O₂ (3%) was also performed. Next, the sections were treated with goat serum (10%) for 20 min and incubated with primary antibodies at 4°C overnight. The secondary antibodies were selected based on the primary antibody host. Following visualization of the signals using the DAB kit, hematoxylin counterstaining was performed. During immunofluorescence (IF) staining, murine articular sections were treated at 97°C for 30 min using 1x sodium citrate, followed by deparaffinization and hydration, and a further 10-min treatment with H₂O₂ (3%) was performed. Then, the sections were treated with goat serum (10%) for 20 min and incubated overnight with primary antibodies at 4°C. After adding fluorescence-conjugated secondary antibodies; the sections were incubated with DAPI dye solution for 10 min at room temperature in the dark. The primary antibody of ACAN (Proteintech), MMP-13 (Proteintech), COL II (Santa), and COL X (Abcam). The sections were microphotographed using an Axiovert 40C optical microscope (Zeiss, Germany), and ImageJ software was used to quantify the percentage of the positive stained area in the total area.