Immunohistochemistry was performed as previously described by our laboratory47 (link)52 (link)53 (link). Primary antibodies against MMP3 (Abcam, San Francisco, CA), MMP13 (Abcam, San Francisco, CA) and type II collagen neoepitopes, exposed when type II collagen is cleaved by MMP13, were used54 (link). Sections were first dewaxed in xylene, and rehydrated through a series of graded ethanol solutions ending in water. Antigen retrieval was performed in 10 mM sodium citrate at 95 °C for fifteen minutes. Sections were blocked in 5% goat serum, then incubated with primary antibodies overnight. Lastly, tissues were incubated in HRP-conjugated secondary antibody and visualized with the substrate DAB (brown precipitate). All tissues were visualized using a Leica DM Series inverted fluorescence/light microscope (Leica Microsystems, Richmond Hill, ON, Canada). At least three pairs of animals were stained for each antibody and multiple sections were used for each trial.
Mmp13
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Canada
MMP13 is a matrix metalloproteinase enzyme that plays a role in the breakdown of extracellular matrix components. It is involved in the remodeling and degradation of the extracellular matrix.
Automatically generated - may contain errors
Lab products found in correlation
Aggrecan, by Abcam (31 mentions)
Adamts5, by Abcam (29 mentions)
Collagen 2, by Abcam (18 mentions)
Gapdh, by Abcam (18 mentions)
Col2a1, by Abcam (16 mentions)
Adamts4, by Abcam (16 mentions)
β actin, by Abcam (16 mentions)
Mmp 9, by Abcam (14 mentions)
Pvdf membrane, by Merck Group (12 mentions)
Bcl 2, by Abcam (11 mentions)
β actin, by Cell Signaling Technology (10 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Tnf α, by Abcam (9 mentions)
Cleaved caspase 3, by Cell Signaling Technology (9 mentions)
Mmp 1, by Abcam (9 mentions)
Cox 2, by Abcam (8 mentions)
Interleukin 6 (il 6), by Abcam (8 mentions)
Runx2, by Abcam (7 mentions)
Gapdh, by Proteintech (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (6 mentions)
185 protocols using mmp13
1
Immunohistochemical Analysis of Cartilage Degradation
2
Chondrocyte Protein Expression Analysis
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Chondrocyte pellets were dissolved in citrate-EDTA buffer (55 mM/5 mM; pH 6.8) for 10 min at 37 °C and centrifuged at 10 K rpm for 15 s. Then, the cell pellet was washed five times with PBS and resuspended in 200 μL of SDS-NuPAGE buffer for NuPAGE gel (Invitrogen, Grand Island, NY, USA) running. Immunoblotting was performed using monoclonal antibodies against Type Ⅰ collagen, Type Ⅱ collagen (1:1000 dilution; EMD Millipore, Billerica, MA, USA), MMP-13 (Abcam, Cambridge, MA, USA) TGF-β (1:1000 mg/mL; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and PPAR-γ (Abcam, Cambridge, MA, USA). The loading control, β-actin, will be detected using anti-β-actin antibody (1:10,000 dilution; Abcam, Cambridge, MA, USA). Secondary antibodies tagged horseradish peroxidase (HRP) (rabbit for TGF-β 1:20,000 dilution and mouse for type Ⅱ collagen and β-actin, 1:5000 dilution) was used. Proteins separated in the gel and the gel were transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using TE77XP semidry blotter with 10 V for 3 h (Hoefer, Inc., Holliston, MA, USA). Protein band signals on blots were detected on Amersham Hyperfilm which was enhanced chemiluminescence (GE Healthcare Life Sciences, Pittsburgh, PA, USA) using SuperSignal West Pico Chemiluminescent Substrate kit (Thermo Fisher Scientific, Rockford, IL, USA).
3
Immunohistochemical Analysis of Osteoarthritis
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The de-paraffinized knee joint sections from DMM and sham mice were incubated overnight at 4° C with primary antibodies against BMP5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Runx2, Col2a, MMP13, Adamts5 (Abcam, Cambridge, UK), as well as p16 and p21 (Proteintech, Rosemont, IL, USA). The slides were then incubated with the secondary antibody (Jackson ImmunoResearch Laboratories, Baltimore Pike, USA, 1:200)) in blocking solution for 1 h at room temperature, and developed with 3,3′-diamino-benzidine (DAB, ZSGB-Bio, Beijing, China).
4
Protein Expression Analysis of Chondrocyte Differentiation
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After harvesting, total proteins were extracted from the cultures. Protein content was assessed by the Pierce BCA assay kit (Biyuntian, China). Total protein (30 µg) was loaded on to 10% SDS polyacrylamide electrophoresis gels and transferred on to a PVDF membrane. The membrane was immunoblotted overnight at 4°C with antibodies against Col2a1 (1:1000, Abcam, U.S.A.), Sox9 (1:200, Sigma–Aldrich), Runx2 (1:500, Sigma–Aldrich), Mmp13 (1:500, Abcam), Pparγ (1:200, Santa Cruz Biotechnology), Pparγ2 (1:500, Elabscience), Col10a1 (1:200, Abcam), Gapdh (1:2000, Transgen), and β-actin (1:4000, Santa Cruz Biotechnology). The membrane was then incubated with secondary anti-mouse or anti-goat IgG antibodies (1:2000, Transgen) conjugated with peroxidase for 60 min at room temperature. The signal was detected by chemiluminescence using the ECL-Plus Detection System (Transgen). Protein semi-quantitation was based on three independent experiments. The densitometric intensities of protein bands were semi-quantitated using Bandscan 5.0 (Glyko Biomedical, U.S.A.) software and values were normalized to those of β-actin or Gapdh for each sample.
5
Immunofluorescence Analysis of Collagen II and MMP13 in NPCs
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NPCs were cultured in 35 mm glass culture dishes and treated with different interventions for 24 h according to the experimental design. NPCs were then washed with cold PBS, fixed in 4% paraformaldehyde for 15–30 min, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with 5% bovine serum albumin (BSA) for 1 h at 37°C. Next, samples were incubated with primary antibodies against collagen II (1 : 100; Abcam, UK) and MMP13 (1 : 50; Abcam, UK) overnight at 4°C. The following day, samples were incubated with Alexa Fluor®488 or 594-labeled goat anti-rabbit IgG antibody (1 : 100) for 1 h at room temperature, then labeled with diamidino-2-phenylindole for 5–10 min. Finally, samples were observed under a confocal fluorescence microscope (Leica, Germany), and the fluorescence intensity was analyzed by Image J software 2.1 (Bethesda, MD, USA).
6
Western Blot for Protein Expression
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After cell treatment or transfection, lysates were collected using RIPA solution containing protease inhibitor cocktail, followed with incubation on ice for 30 min and centrifugation at 12,000 ×g for 15 min at 4°C. Supernatant was collected, quantified using the BCA protein quantification kit (Thermo, USA), and denatured at 99°C for 5 min. Protein samples with equal amount of 30 μg were separated by 10% SDS-PAGE, then transferred to the PVDF membrane, and incubated with blocking solution and later with the primary antibody of anti-DICER1 (Abcam, USA), TLR3 (Biosen, China), COX2 (Abcam, USA), MMP3 (Abcam, USA), MMP8 (Proteintech, USA), MMP13 (Abcam, USA), and GAPDH (Proteintech, USA), respectively, at 4°C overnight. The membranes were washed with TBST, incubated with the secondary antibody (labeled with HRP) at room temperature for 1 h, and washed again before developed by using the SuperSignal® ECL West Pico kit (Thermo Scientific, USA) and captured by using the Syngene Image system, and the specific bands were scanned for density using Genesys softwares. The intensity of specific binding bands was calculated against the endogenous control (GAPDH), and data were showed as fold change against the control.
7
Investigating Cellular Mechanisms via Pharmacological Inhibition
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NECA and the A2aR antagonist SCH 58261 were obtained from Sigma (St. Louis, MO). The mTOR antagonist OSI-027 was purchased from MedChem Express (Monmouth Junction, NJ). Purified anti-rabbit A2bR polyclonal antibody was purchased from Bioss Antibodies (Edinburgh, UK). Purified anti-rabbit MMP-2, MMP-7, MMP-9, MMP-13, phosphorylated (p)-mTOR, p-PI3K, p-AKT, A2aR, and FITC-IgG (fluorescein isothiocyanate–immunoglobulin G) fluorescent secondary antibody were purchased from Abcam (Cambridge, UK). The EMT Antibody Sampler Kit was purchased from Cell Signaling Technology (Danvers, MA).
8
Recombinant Rat IL-1β and EZH2 Pathway
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Recombinant rat IL-1β was obtained from Peprotech (Rocky Hill, NJ, USA). EPZ005687 was obtained from MedChemExpress (1396772-26-1). Antibodies against EZH2 and Sox-9 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against H3K27me3 and normal rabbit IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against β-actin, aggrecan, Collagen II, ADAMTS5 and MMP13 were purchased from Abcam (Cambridge, MA, USA).
9
Protein Expression Analysis of Wound Healing
10
Evaluating Cartilage Markers in Osteoarthritis
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We performed immunohistochemical staining to detect the expression of phosphor-mammalian target of rapamycin (p-mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) in the articular cartilage. Rabbit polyclonal antibodies to p-mTOR, LC3 (Cell Signaling, Beverly, MA, USA), VEGF, MMP13 (Abcam, Cambridge, MA, USA), and COL10A1 (Abbiotec, San Diego, CA, USA) were used at a 1:100 dilution and incubated overnight at 4°C. Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Molecular Probes, Grand Island, NY, USA) was used at 1:200 dilution as the secondary antibody against the primary antibodies at room temperature for 2 hours. After staining, we evaluated the expression levels in the articular cartilage using Northern Eclipse software (Empix Imaging Inc, Cheektowaga, NY, USA).
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