Arg55241

Fresh tarsal plates and explants were fixed in 4% paraformaldehyde for 1 hour and dehydrated with a series of sucrose before embedded in O.C.T. (4583; SAKURA, Chiba Prefecture, Japan) for sectioning (10 µm).
The sections as well as fixed cells were blocked in 10% donkey serum (ANT051; AntGene, China) for 1 hour, and incubated with rabbit polyclonal antibodies against keratin14 (Krt14; 1:500, ab181595; Abcam, UK), Krt6 (1:300, 905701; BioLegend, San Diego, CA), Krt1 (1:300, 905601’ BioLegend), and peroxisome proliferator-activated receptor γ (PPARγ; 1:100, ARG55241; Arigo) overnight at 4°C. Then samples were stained by Alexa Fluor 488 donkey anti-rabbit IgG (1:200, ANT024; AntGene) for 1 hour. DAPI (10236276001; Roche, Germany) and Nile Red (72485; Sigma-Aldrich) were used for nucleus and lipid staining, respectively. Images were obtained with a laser scanning confocal microscope (Nikon, Japan).

For 14 days, the knee joints were decalcified in 14% ethylenediamine tetraacetic acid solution after μCT analysis, embedded in paraffin and prepared as 4 μm thick sections as described in the literature.25 (link) The paraffin sections were stained with Toluidine Blue staining (TB) and Alcian Blue Hematoxylin/Orange G staining (ABH) to determine the tissue structure of the knee joint. The thickness and wear degree of knee cartilage were analyzed using OsteoMetrics software (Decatur, GA, USA). Expressions of Collagen Type II (Col2), matrix metalloproteinase 13 (MMP13), Aggrecan, and peroxisome proliferator-activated receptor gamma (PPARG) were observed using immunohistochemistry. Immunohistochemical staining was performed regarding previous literature reports.26 (link) Anti-Col2 (Abcam, ab34712, 1:200), anti-MMP13 (Abcam, ab39012, 1:100), anti-Aggrecan (Bioss, bs-11655R, 1:200) and anti-PPARG (Arigo, ARG55241, 1:200) were utilized in this study. MMP13 and PPARG expression were counted by the percentage of positively stained cells to total chondrocytes in the region of interest. By counting the positive stained area in the region of interest, Col2 expression was quantified.