Ab41037

Paraffin sections were deparaffinized in xylene for immunohistochemical staining in cartilage tissue. Next, the sections were washed and, and hydrated with ethanol. In order to expose the Antigen of the sample, 0.1% trypsin was treated for 30 min. Sections were stained via the LSAB2 Horseradish Peroxidase Kit (Dako, Santa Clara, CA, USA) according to the manufacturer’s instructions. sections of the slide were incubated with an antibody against Adamts5 (ab41037, Abcam, Cambridge, UK) for 12 h at room temperature, followed by visualization of immunoreactive proteins using DACO AEC + high sensitivity matrix chromosome solution (Dako, Santa Clara, CA, USA).

After the treatments, the cells were lysed on ice for 20 min with a mixture of 100 μl RIPA buffer (Fdbio science, Hangzhou, China), 1 μl protease inhibitor (Fdbio science) and 1 μl phosphatase inhibitor (Fdbio science) and centrifuged at 14,000 g for 25 min. Protein concentrations were determined using a BCA Total Protein Assay Kit (CWBIO, China). Protein samples (40 μg/lane) were separated on SDS-PAGE gels (8% or 12%, Epizyme, China) and electrotransferred to PVDF membranes. The blots were incubated with the following primary antibodies: anti-NLRP3 (1:500, A12694, ABclonal), anti-GSDMD (1:500, 39754, CST), anti-p20 (1:500, AF4005, Affinity), anti-CHOP (1:500, ab11419, Abcam), anti-GRP78 (1:500, ab21685, Abcam), anti-COL2A (1:500, ab34712, Abcam), anti-Aggrecan (1:500, ab36861, Abcam), anti-MMP3 (1:500, ab52915, Abcam), anti-MMP13 (1:500, ab39012, Abcam), anti-ADAMTS5 (1:1000, ab41037, Abcam), and anti-SREBP1 (1:1000, ab28481, Abcam). The membranes were washed and incubated with the HRP-conjugated secondary antibodies (1:8000, ABclonal, China) for 1 h. The bands were developed with chemiluminescence reagents and imaged by a myECL imager (Syngene G:BOX ChemiXT4, United Kingdom). The experiment was repeated in triplicate, and the blots were quantified by ImageJ software. An antibody against β-actin (AC026, 1:1000; ABclonal) served as an endogenous control.

Immunofluorescence staining was performed on the sagittal sections of the murine knee joint to estimate the severity of OA. The incubation of primary antibody against MMP13 (rabbit, cat. no. #18165-1-AP; Proteintech) and collagen II (rabbit, cat. no. #ab34712; Abcam) on the slices was administered after antigen retrieval and blocking. Then, the sections were probed with fluorescein isothiocyanate–conjugated secondary antibodies (Alexa Fluor 555-labeled Donkey Anti-Rabbit IgG (H + L) and Alexa Fluor 488-labeled Donkey Anti-Rabbit IgG (H + L), Beyotime Institute of Biotechnology). The nuclei were counterstained with DAPI (Sigma, D9542). The images were viewed using a Nikon A1 confocal microscope and analyzed using ImageJ software.
For immunohistochemistry, the knee slices were prepared as described in the histological analysis and subsequently subjected to antibodies against MMP3 (rabbit, cat. no. #ab52915; Abcam) and ADAMTS5 (rabbit, cat. no. #ab41037; Abcam). Percentages of positive MMP3 and ADAMTS5 chondrocytes were determined by counting the number of immunostained cells and dividing by the total number of chondrocytes visualized by a hematoxylin counterstain.