Csa 2 biotin free catalyzed amplification system

Tissue samples were fixed in 4% paraformaldehyde buffered with PBS at 4 °C for 1 day, decalcified with 10% EDTA (pH 7.4) at 4 °C for 2 weeks and embedded in paraffin. Sagittal sections (4-μm thick) were cut from specimens. For immunofluorescence, sections were incubated with antibodies against ADAMTS5 (1:50; D-16, Santa Cruz Biotechnology, TX, USA), type II collagen (1:200; LB-1297, LSL, Tokyo, Japan), type X collagen (1:200; LB-0092, LSL, Tokyo, Japan), Hic-5 (1:200; 611165, BD Biosciences, NJ, USA) and MMP13 (1:100; 18165–1-AP, Proteintech, IL, USA) diluted in the blocking reagent. For the staining of ADAMTS5, MMP13, type II collagen and type X collagen we used the CSA II Biotin-Free Catalyzed Amplification System (Agilent Technologies, CA, USA) and DAPI. Hic-5 staining in chondrocytes was performed by the same procedure as immunofluorescence. At least three samples were tested in each assay.

Histological analyses. Tissue samples were fixed in 4% paraformaldehyde buffered with phosphate-buffered saline (PBS, pH 7.4) at 4℃ for 1 day. Specimens were decalcified with 10% EDTA (pH 7.4) at 4℃ for 4 weeks, embedded in paraffin, and 6-µm thick sagittal sections were cut from specimens. Safranin O staining was performed according to standard protocols. For immunohistochemistry, sections were incubated with antibodies against MMP13 (1:200, abcam, cat. no. ab219620), p-EGFR (1:300, abcam, cat. no. ab40815). For immunofluorescence, a CSA II Biotin-Free Catalyzed Amplification System (Agilent Technologies) and Hoechst 33,258 (Agilent Technologies) counterstain were used.
TUNEL staining was performed with an In Situ Cell Death Detection Kit (Roche, Inc, cat. no. 11684817910)according to the manufacturer’s instructions. Histological analyses were performed at least three times using per group for confirmation of results. Images were visualized under a fluorescence microscope (Keyence, Osaka, BZ-X710). Counts of immunofluorescence-positive cells were calculated in five independent squares (100 × 100 µm) of articular cartilage harvested from the mouse medial tibial plateau, using BZ analyser software (Keyence). The same parameters were used for all acquisitions, and representative pictures are shown in the figures.