IL-1β (Pepro Tech, USA), Forskolin (APExBIO Technology, USA), SFN (APExBIO Technology, USA), Rat IL-1β ELISA Kit (Elabscience, China), Rat TNF-α ELISA Kit (Elabscience, China). For immunohistochemical and immunofluorescence analyses, the following antibodies were used: anti-BMAL1 (Abcam, #ab3350), anti-NRF2 (Proteintech, #16396-1-AP), anti-Aggrecan (Proteintech, #13880-1-AP), anti-MMP13 (Abcam, #ab39012), anti-p65 (Santa Cruz Biotechnology, #(F-6): sc-8008), anti-Phospho-p65(Santa Cruz Biotechnology, #(A-8): sc-166748), and GAPDH (Proteintech, #60004-1-lg). For immunohistochemical and immunofluorescence analyses, the following antibodies were used: anti-BMAL1 (Abcam, #ab3350), anti-NRF2 (Proteintech, #16396-1-AP), anti-Aggrecan (Proteintech, #13880-1-AP), anti-MMP13 (Abcam, #ab39012), and anti-p65(Invitrogen, PA5-16545).
Anti mmp13
Manufactured by Abcam
Sourced in United Kingdom, United States, China
Anti-MMP13 is a laboratory reagent used to detect and measure the levels of matrix metalloproteinase 13 (MMP13) in various biological samples. MMP13 is an enzyme involved in the breakdown of extracellular matrix proteins and plays a role in various physiological and pathological processes. This product can be used as a tool for research purposes to study the expression and function of MMP13.
Automatically generated - may contain errors
Lab products found in correlation
Anti collagen 2, by Abcam (6 mentions)
Anti adamts5, by Abcam (6 mentions)
Anti gapdh, by Abcam (5 mentions)
Anti mmp 3, by Abcam (4 mentions)
Anti colii, by Abcam (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Anti adamts4, by Abcam (3 mentions)
Anti gapdh, by Proteintech (3 mentions)
Anti col2a1, by Abcam (3 mentions)
Anti rabbit igg, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Anti α sma, by Abcam (3 mentions)
Anti mmp2, by Abcam (2 mentions)
Anti nlrp3, by Proteintech (2 mentions)
Bzatp, by Merck Group (2 mentions)
Anti p2x7, by Abcam (2 mentions)
Anti caspase 1, by Proteintech (2 mentions)
Anti p21, by Abcam (2 mentions)
Anti runx2, by Abcam (2 mentions)
Anti sox9, by Abcam (2 mentions)
48 protocols using anti mmp13
1
Inflammatory Response Modulation: A Comprehensive Protocol
2
Immunoblot Analysis of ECM Proteins
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Immunoblot procedures were performed as described previously [16 (link)]. As primary antibodies, mouse anti-α-SMA (2 μg/mL; Invitrogen), anti-GADPH (0.05 μg/mL; Sigma-Aldrich), rabbit anti-collagen I (1:1000; Abcam, Cambridge, UK; cat. no. ab34710), rabbit anti-collagen III (1:5000; Abcam; cat. no. ab7778), rabbit anti-fibronectin (1:1000; Abcam; cat. no. 1b45688), anti-matrix metalloproteinase (MMP)-1 (1:1000; Abcam; cat. no. ab134184), and anti-MMP-13 (1:1000; Abcam; cat. no. ab51072) were used. Anti-mouse IgG (1:50000; Sigma-Aldrich) and anti-rabbit IgG (1:10000 Sigma-Aldrich) were used as secondary antibodies. Photographs were acquired with an Amersham Imager 600 (GE Healthcare). Densitometric analysis was performed using Image Quant TL software. Expression of proteins of interest was normalized to the expression of GADPH.
3
Immunohistochemical Analysis of Cartilage Markers
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As previously reported [11 (link)], cell samples were fixed in 44% paraformaldehyde for 10 min and washed with distilled water. Next, the cells were treated with 3% H2O2 for 15 min at room temperature to eliminate the endogenous peroxidase activity and blocked with normal goat serum for 40 min at room temperature. Then, the cells were incubated with anti-Col2 (Abcam, UK; 1:200), anti-ColX (Santa Cruz Biotechnology; 1:200), and anti-MMP13 (Abcam, UK; 1:200) primary antibodies overnight at 4 °C. The sections were subsequently incubated with a secondary antibody, IgG-HRP (CST; 1:200). The resulting sections were photographed under a microscope.
4
Protein Expression Analysis in Intervertebral Discs
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Based on a previous report, proteins were measured through western blotting [15 (link)]. For the isolation of proteins in the tissues, nucleus pulposus tissues were first ground in chilled mortar in the presence of liquid nitrogen. Immediately after grinding, the tissue powder was lysed with radioimmunoprecipitation assay (RIPA) buffer. Total proteins were then separated on polyacrylamide gels and transferred to a polyvinylidene difluoride membrane before being probed overnight at 4°C with antibodies (anticleaved-caspase-3, anticleaved-caspase-8, anti-cleaved-caspase-9, anti-MMP-1, anti-MMP-2, anti-MMP-3, anti-MMP-13, and anti-ADAMTS-4; Abcam, Cambridge, UK), then washed three times with PBST. Then, membranes were treated with the corresponding secondary antibodies. Finally, the membrane was processed using an ECL kit for color reaction. Western blotting results were normalized to those of GAPDH for quantification.
5
Investigating Molecular Pathways in Osteoarthritis
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The antibodies used in this study were as follows: anti-P2X7 (Abcam, Cambridge, UK; cat. no. ab109054), anti-collagen II (Abcam; cat. no. ab34712), anti-MMP13 (Abcam; cat. no. ab39012), anti-AMPKα1 (Abcam; cat. no. ab32047), anti-mTOR (Abcam; cat. no. ab109268), anti-NLRP3 (Proteintech; cat. no. 19771-1-AP), anti-caspase-1 (Proteintech; cat. no. 22915-1-AP), anti-LC3B (Abcam; cat. no. ab192890), anti-Beclin-1 (Abcam; cat. no. ab62557), anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Proteintech; cat. no. 10494-1-AP), and horseradish peroxidase (HRP)-labeled IgG (Beyotime; cat. no. A0208). The reagents used in the experiment were as follows: MIA (Sigma, St. Louis, MO, USA; cat. no. I2512), the P2X7 receptor agonist BzATP (Sigma; cat. no. B6396), the mTOR activator MHY1485 (Sigma; cat. no. SML0810), the mTOR inhibitor rapamycin (Sigma; cat. no. V900930), the NLRP3 inhibitor CY-09 (Sigma; cat. no. SML2465), the AMPK activator A-769662 (Sigma; cat. no. SML2578), and the AMPK inhibitor compound C (Sigma; cat. no. P5499). The concentrations, dosages, and preparation of the reagents were described previously [43 (link), 45 (link)].
6
Western Blot Analysis of Cartilage Extracellular Matrix Proteins
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The total protein was extracted using RIPA reagent (Beyotime), and the protein concentration was detected using BCA detection kit (Beyotime). 40 μg protein sample/lane was subjected to 10% SDS-PAGE and then transferred to PVDF membranes. The membranes were sealed with 5% skimmed milk at room temperature and then incubated at 4℃ overnight with the corresponding primary antibody. Following that the membranes were incubated for 2 h at room temperature with the corresponding secondary antibody. Protein bands were developed using ECL (Millipore), and images were taken and analyzed using Bio-Rad ChemiDoc XRS + (Bio-Rad, CA, USA). Primary antibodies (Anti-GJA1 (1:1000), anti-Collagen II (1:1000), anti-MMP-3 (1:2000), anti-MMP13 (1:2000), anti-ADAMTS4 (1:1000), anti-ADAMTS5 (1:1000), anti-GAPDH (1:5000)) and HRP labeled secondary antibodies (1:5000) were obtained from Abcam (Cambridge, USA). GAPDH was the normalization.
7
Protein Expression Analysis via Western Blotting
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We performed western blotting (WB) according to standard procedures. The following primary antibodies were used: anti-Hnrnpk (Abcam 39975, 1:5000), anti-Bax (Cell Signaling Technology 14796, 1:1000), anti-p21 (Abcam 109520, 1:1000), anti-Ihh (Abcam 52919, 1:1000), anti-Runx2 (Abcam 192256, 1:1000), anti-Mmp13 (Abcam 219620, 1:500), anti-Sox9 (Abcam 185966, 1:1000), anti-Hif1α (Cell Signaling Technology 36169, 1:250), anti-Gapdh (Proteintech 60004-1, 1:2000), anti-Pfkfb3 (Abcam 181861, 1:500), anti-Ldha (Proteintech 19987, 1:2000), and anti-β-Actin (Affinity AF7018, 1:2000). The following secondary antibodies were used: goat anti-rabbit IgG H&L (Abcam ab205718, 1:2000) and goat anti-mouse IgG H&L (Abcam ab205719, 1:2000). The Densitometry analysis of WB was exerted using software Image J (v 1.51). The original western blots were shown in the Supplementary material Western blots .
8
Histological Analysis of Cartilage
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Samples of paraffin-embedded tissue were cut to yield 4-µm thick sagittal sections which were then subjected to Hematoxylin and Eosin (HE), Masson’s trichrome and Safranin O-Fast Green staining. Immunohistochemical staining for matrix metalloproteinase (MMP)-13 and alpha-smooth muscle actin (α-SMA) were conducted by deparaffinizing tissue sections, rehydrating them with an ethanol gradient, and conducting antigen retrieval by processing samples for 30 min with citrate buffer. Samples were then washed with PBS, treated with 3% H2O2 to quench endogenous peroxidase activity, blocked for 1 h in 10% sheep serum at 37°C, and treated overnight with anti-MMP13 (1/500, Abcam, USA) or anti-α-SMA (1/200, Abcam) at 4°C. Sections were then probed with an appropriate HRP-linked secondary antibody (Abcam) for 45 min, washed with PBS, stained with the DAB substrate solution, and counterstained for 30 s with hematoxylin. A light microscope (Olympus Optical, Tokyo, Japan) was then used to image sections, which were evaluated using ImageJ. Two investigators blinded to experimental conditions calculated the percentage of staining positive area in six randomly selected fields of view for each articular cartilage section at a magnification of ×400.
9
Western Blot Analysis of Chondrocyte Markers
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Radioimmunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China) was used to isolate total proteins in cells, and protein concentration was quantified by a NanoDrop 3000 (Thermo Fisher Scientific). sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was used to separate proteins, and then proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. After that, membranes were blocked in skim milk for 2 h at 37°C and then incubated with primary antibody (Ab) at 4°C overnight. Following 2-h incubation with secondary Ab marked with horseradish peroxidase (HRP) (1:2000; Cell Signaling Technology, Danvers, MA, USA). The chemiluminescence was developed using an Enhanced chemiluminescence (ECL) detection kit (Beyotime). The primary Abs were as follows: anti-ADAMTS5 (1:1000; Abcam, Cambridge, UK), anti-MMP13 (1:1000; Abcam, Cambridge, UK), anti-COL2A1 (1:1000; Abcam, Cambridge, UK), anti-proliferating cell nuclear antigen (PCNA) (1:1000; Abcam, Cambridge, UK), anti-β-actin (1:4000; Cell Signaling Technology, Danvers, MA, USA), and anti-cleaved caspase-3 (anti-C-caspase3; 1:1000; Cell Signaling Technology).
10
Protein Expression Analysis in Cells
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Cells were lysed in RIPA buffer with a protease inhibitor cocktail (KeyGEN, Jiangsu, China) to obtain protein lysates, which were then quantified with a BCA quantification kit (Beyotime, Shanghai, China). After SDS‒PAGE electrophoresis, the proteins were transferred to PVDF membranes (Millipore, Massachusetts, USA) and blocked in WB blocking solution at room temperature for 1 h (Thermo Fisher Scientific, USA), followed by incubation with specific antibodies such as anti-ADAMTS4 (#11865-1-AP, Proteintech, USA), anti-ADAMTS5 (#14351, Cell Signaling Technology, USA), anti-MMP13 (#ab219620, Abcam, UK), anti-WTAP (#60188-1-Ig, Proteintech, USA), anti-FRZB (#ab205284, Abcam, UK), anti-β-catenin (#8814S, Cell Signaling Technology, USA) and β-actin (#81115-1-RR, Proteintech, USA) at 4 °C overnight. After washing with 1% TBST for 30 min and incubation with secondary antibodies (1:5000, Abcam) for 2 h, the signal on the membranes was detected by a chemiluminescence system (Tanon 5200 Multi).
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