Mmp 13 antibody

The rats were sacrificed and the knee joints were collected and fixed in 4% paraformaldehyde for 48 h, then decalcified in 10% ethylenediaminetetraacetate (EDTA) for 4 weeks. After serial dehydration, the joints were embedded in paraffin and sagittally sectioned at 3 μm thickness. The sections were de-waxed and stained with hematoxylin–eosin (HE, Jian Cheng Biotech, China) and safranin O/fast green (Solarbio, Beijing, China). Then, three independent observers graded the sections based on the scoring criteria reported by Osteoarthritis Research Society International (OARSI) [45 (link)].
The immunohistochemical staining was used to analyze the secretion of MMP-13. The dewaxed sections in the different groups were washed with PBS and exposed to 3% (v/v) hydrogen peroxide H₂O₂ to block endogenous peroxidase activity for 15 min at room temperature. After blocking with normal goat serum for 20 min at room temperature, primary MMP-13 antibodies were added (1:200 dilution, Abcam, USA) and incubated at 4 °C overnight. The sections were incubated with secondary antibody for 15 min and then added with biotin-labeled horse radish peroxidase for 15 min. A 3, 3′-diaminobenzidine tetrahydrochloride (DAB) kit and hematoxylin were used for color development and nuclei dye. Tissues sections were observed and photographed with a microscope.

Immunofluorescence experiments were carious out according to previously described [49 (link)]. Chondrocytes from various treatment groups were washed twice in PBS before being used to make cell smears. The cells were fixed for 30 minutes at room temperature with 4 percent paraformaldehyde, washed twice with PBS, and then treated for 15 minutes at room temperature with 0.1% Triton-X 100 to render them permeable. After washing the permeate, Collagen II (1:200 dilution, Abcam, UK) and MMP-13 antibody (1:200 dilution, Abcam, UK) were added and the reaction was incubated overnight at 4°C. After washing out the first antibody, a fluorescently labeled secondary antibody (1:200, Abcam, UK) was added and incubated at room temperature for 1 hour before being washed three times with PBS. After adding DAPI staining and washing out the staining solution the cells were observed under the fluorescence microscope (Olympus DP80, Japan).