Whole cell protein extraction was developed using a lysis buffer. Electrophoresis and blotting procedures have been described previously13 (link). Immunoblots were incubated with the appropriate antibody (anti-phospho p38 diluted 1:1000, Millipore, MA, USA; anti-p38 diluted 1:1000, Millipore, MA, USA; anti IκBα diluted 1:1000, Cell Signaling Technology, MA, USA; anti-MMP-13 diluted 1:500, Santa Cruz Biotechnology, CA, USA; anti-NOS2 diluted 1:1000, Cell Signaling Technology, MA, USA; anti-COX-2 diluted 1:1000, Dako, Denmark) and visualized using an Immobilon Western kit (Millipore, MA, USA) and anti-rabbit (GE Healthcare, UK) horseradish-peroxidise-labelled secondary antibody diluted 1:2000. To confirm equal loading for each sample, after stripping in glycine buffer at pH3, membranes were reblotted with anti-β-actin antibody diluted 1:5000 (Sigma, MO, USA). Autoradiographs were analyzed with an EC3 imaging system (UVP, CA, USA).
Anti mmp 13
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-MMP-13 is a laboratory reagent used for the detection and quantification of Matrix Metalloproteinase-13 (MMP-13) in various biological samples. MMP-13 is an enzyme involved in the breakdown of extracellular matrix components and plays a role in tissue remodeling and degradation. This product can be used in research applications that require the measurement or analysis of MMP-13 levels.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd9, by System Biosciences (3 mentions)
Ec3 imaging system, by Analytik Jena (2 mentions)
Anti rabbit, by GE Healthcare (2 mentions)
Anti nos2, by Cell Signaling Technology (2 mentions)
Anti mmp 9, by Santa Cruz Biotechnology (2 mentions)
Anti mmp 1, by Santa Cruz Biotechnology (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Anti phospho p38, by Merck Group (1 mentions)
Anti p38, by Merck Group (1 mentions)
Immobilon western kit, by Merck Group (1 mentions)
Anti iκbα, by Cell Signaling Technology (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Vectashield mounting medium with dapi, by Vector Laboratories (1 mentions)
Anti vcam 1, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Anti pgrn, by Santa Cruz Biotechnology (1 mentions)
Immobilon western detection kit, by Merck Group (1 mentions)
Anti cox 2, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole dapi, by Abcam (1 mentions)
9 protocols using anti mmp 13
1
Whole Cell Protein Extraction and Immunoblotting
2
Immunofluorescent Analysis of MMP-13 Expression
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Cells were washed, fixed with 4% paraformaldehyde, and incubated in 5% normal horse serum in 0.2% Triton PBS for 1 h. A rabbit polyclonal anti-MMP-13 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the cells overnight at 4°C. The cells were further incubated with a secondary antibody, CFL 488-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology), for 2 h. The cells were then washed and mounted with Vectashield mounting medium with DAPI (Vector Laboratories Inc., Burlingame, CA, USA). The stained cells were examined using an Olympus BX51 microscope, using a 40× objective and software (Olympus). To examine the protein distribution, the proportion of MMP-13 localizing with or without DAPI in the cells was quantified using ImageJ ver. 1.48 (NIH, Bethesda, MD, USA).
3
Protein Extraction and Western Blot Analysis
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After the cell treatment, cells were rapidly washed with ice-cold phosphate buffered saline and scraped in lysis buffer for protein extraction (10 mM Tris/HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM Sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 0.5% Triton X-100, 1 mM PMSF and protease inhibitor cocktail from Thermo Scientific). Lysed cells were centrifuged at 14.000 g for 20 min. SDS-PAGE and blotting procedure were carried on as previously described38 (link). Immunoblots were incubated with the appropriate antibody (anti-PGRN diluted 1:1000, Santa Cruz, CA, USA; anti-NOS2 diluted 1:1000, Cell Signalling, MA, USA; anti-COX2 diluted 1:50, anti-VCAM-1 diluted 1:1000, Cell Signalling, MA, USA; anti-MMP13 diluted 1:500, Santa Cruz, CA, USA) and visualized with an Immobilon Western Detection kit (Millipore, MA) using anti-rabbit (GE Healthcare, UK) horseradish-peroxidise-labelled secondary antibody diluted 1:2000. To confirm equal loading in each sample, the membranes were stripped in stripping buffer (100 mM β-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl pH 6.7) and re-blotted with anti-GAPDH antibody diluted 1:30000 (Sigma, MO, USA). The images were captured and analyzed with an EC3 imaging system (UVP). Densitrometric analyses were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
4
Osteoarthritis Assessment via Safranin O and IF
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Safranin O staining was performed using a commercial staining kit (Solarbio) according to the manufacturer’s instructions. The OARSI grading system was used to evaluate the sections [18 (link)]. For immunofluorescence (IF) staining, prepared sections were incubated with anti-MMP13 (1:100 dilution, SantaCruz Biotechnology) and anti-GEM(1:100 dilution, SantaCruz Biotechnology) primary antibodies. Alexa Fluor 488-conjugated antibodies (Invitrogen) were used as secondary antibodies. All samples were counterstained with 4′,6-diamidino-2-phenylindole (DAPI, Abcam). A BX63 confocal microscope (Olympus) was used to perform imaging. Images were quantified by ImageJ software (NIH).
5
Cytokine-Mediated Chondrocyte Regulation
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Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) Media is obtained from Hyclone (Utah, USA). Penicillin, streptomycin and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY, USA). Recombinant human and mouse IL-1β were obtained from R&D Systems (Minneapolis, MN). The antibodies used in this study are as follow: anti-sirt6 from Abcam (Cambridge, MA); anti-NF-κB p65 and anti-IKb-α from Cell Signaling Technology (Danvers, MA); anti-actin from Sigma; anti-collogan II from Chemicon (Temecula, CA); anti-MMP-13 from Santa Cruz Biotechnology (Santa Cruz, CA); Alexa-Fluor-488- and Alexa-Fluor-545-tagged second antibodies were from Molecular Probes (Eugene, OR); secondary antibodies goat anti-rabbit IRDye 800CW and goat anti-mouse IRDye 680 were from LI-COR Biosciences (Lincoln, NE).
6
Western Blot Analysis of MMPs
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Western blot assays were carried out as previously described [25 (link)]. The protein from primary human chondrocytes was isolated after 24 h of treatment. The primary antibodies used were rabbit polyclonal anti-MMP1 (1:500), anti-MMP2 (1:500), anti-MMP13 (1:500), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1000), goat polyclonal anti-MMP3 (1:500), mouse monoclonal anti-MMP9 (1:1000; Santa Cruz Biotechnology, Inc.), and anti-SRY-related high mobility group-box gene9 (SOX9) (1:2000; Millipore).
7
Western Blot Analysis of Chondrocyte Markers
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Cell pellets were lysed and quantified as previously reported56 (link). The samples (10–30 μg of protein) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma) and transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia, Escondido, CA, USA). Briefly, after blocking with 5% skim milk, membranes were incubated with primary anti-SOX9 (1:3000, Millipore, Billerica, MA, USA), anti-RUNX2, anti-P16 (1:3000, Abcam, Cambridge, UK), anti-COL2A1, anti-AGGRECAN, anti-OCT4, anti-MMP13, anti-COL1A1, anti-P21, anti-P53 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-NANOG (1:1000, BD biosciences, San Jose, CA, USA), anti-SOX2 (1:1000, Cell signaling Technology, Inc., Danver MA, USA), anti-HSP90 or anti-β-ACTIN (1:1,000, Santa Cruz Biotechnology) overnight at 4 °C. Then, membranes were incubated with secondary HRP-conjugated antibodies (1:5000, Santa Cruz Biotechnology) for 1 h at room temperature.
8
Biochemical Profiling of 143B EMV Cargo
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To determine the biochemical composition of the 143B EMV cargo, Western blot analyses were performed according to the previously described method [30] (link). 143B EMVs were homogenized in Tris lysis buffer (20 mM Tris, 137 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM PMSF, and 1 mM DTT). Crude lysates of 143B cells (12.5-25 μg) and EMVs (25-40 μg) were denatured in sodium dodecyl sulfate sample buffer, electrophoresed on 12% denaturing polyacrylamide gels, and visualized by Ponceau stain. For immunoblot analysis, the proteins from the gel were transferred on to a polyvinylidene fluoride (PVDF) membrane and incubated with the following primary antibodies: anti–MMP-1 and anti–MMP-13 (Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); 200 μg/ml each) at 1:200, anti-CD-9 (SBI: System Biosciences (Mountain View, CA, USA); 0.25 mg/ml) at 1:1000, and anti-RANKL and anti–TGF-β (GeneTex (Irvine, CA, USA); 1 mg/ml) at 1:1000 dilution. Detection of the immunostained bands was done by ECL chemiluminescence detection system (Thermo Scientific, Rockford, IL). Image acquisition was done using LabWorks Image Acquisition and Analysis Software 4.6.00.0 (UVP Bioimaging Systems, Upland, CA) and Image Lab software for the ChemiDoc MP system (Bio-Rad Laboratories) at incremental exposure time frame of 15, 30, 60, 180, and 300 seconds.
9
Histochemistry and Immunohistochemistry Protocol
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Evans Blue, triphenyl tetrazolium chloride, hematoxylin-eosin, sirius red, Triton X-100, calcium chloride, zinc chloride, and acetic acid were from Merck (Madrid, Spain). The Masson trichrome staining kit, anti-EMMPRIN, anti-CD68, HRP/DAB IHC kit, and HRP-conjugated secondary antibodies were from Abcam (Cambridge, United Kingdom). Anti-MMP-9 and anti-MMP-13 were from Santa Cruz (Heidelberg, Germany). Ketamine was from Pfizer (New York, NY); the isoflurane was from Abbvie (North Chicago, IL); the propofol was from Fresenius (Bad Homburg, Germany); the fentanyl was from Kern Pharma (Madrid, Spain); the diazepam was from Roche (Basel, Switzerland); and the amiodarone was from Sanofi Aventis (Gentilly, France).
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