Ab182563

Manufactured by Abcam

Sourced in United States

Ab182563 is a laboratory equipment product. It is a high-quality tool designed for scientific research and testing purposes. The core function of this product is to facilitate the execution of specific laboratory procedures and experiments. No further details are available without the risk of potential bias or extrapolation.

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Lab products found in correlation

7 protocols using ab182563

Histological and Immunohistochemical Analysis of Cartilage and Angiogenesis

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The samples were fixed with 4 % PFD, decalcified 15 % EDTA solution, dehydrated and embedded in paraffin for sectioning (4.5 μm). The sections (transverse slice direction of the central portion within the segmental radial defect area; sagittal slice direction of the central part within the mandibular defect area) were stained with safranin O/Fast Green for the cartilage matrix. After deparaffinization and microwave radiation antigen repair, immunohistochemistry was carried out using primary antibodies against Col I (abcam, ab6308), Col II (abcam, ab34712), Col X (abcam, ab182563) and CD31 (abcam, ab182981), an HRP-conjugated anti-rabbit secondary antibody and 3,3′-diaminobenzidine (DAB, brown staining indicates positive immunostaining). The antibody information used in this study was listed in Table S1. Typically, there were six slice samples performed for histological/IHC staining and their quantification in small animal models (mouse ectopic model and rat femoral defect model), with one representative image illustrated. For large animal models (dog segmental radial defect model and dogs mandibular defect model), three slice samples for histological/IHC staining and their quantification, with one representative image presented. All staining slides were captured using a light microscope (Nikon E100, Japan).

Sun L., Niu H., Wu Y., Dong S., Li X., Kim B.Y., Liu C., Ma Y., Jiang W, & Yuan Y. (2024). Bio-integrated scaffold facilitates large bone regeneration dominated by endochondral ossification. Bioactive Materials, 35, 208-227.

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Protein Extraction and Analysis from BMSCs and Cartilage

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RIPA buffer (Beyotime, Shanghai, China) was utilized for extracting total proteins from BMSCs and murine cartilage tissues. Quantification of proteins were achieved using a Bicinchoninic acid Protein Assay Kit (Beyotime). Protein samples (20 μg) were resolved with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Invitrogen) [29 (link)]. After blocked with 5% nonfat milk, the membranes were incubated with primary antibodies against Map3k2 (ab33918, 1:10000, Abcam), Aggrecan (13880-1-AP, 1:1000, Proteintech, Chicago, USA), collagen II (Col2, ab34712, 1:1000, Abcam), collagen X (Col10, ab182563, 1:1000, Abcam), matrix metalloproteinase 13 (MMP13, ab39012, 1:3000, Abcam), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS, ab41037, 1:250) and β-actin (ab6276, 1:5000, Abcam) overnight at 4°C. After that, the membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Abcam) at room temperature for 2 hours. Eventually, protein signals were visualized using an enhanced chemiluminescence kit (Cwbiotech, Beijing, China) with an Odyssey infrared imaging system (LI-COR, Lincoln, NE, USA).

Liu J., Tang G., Liu W., Zhou Y., Fan C, & Zhang W. (2022). MiR-20a-5p facilitates cartilage repair in osteoarthritis via suppressing mitogen-activated protein kinase kinase kinase 2. Bioengineered, 13(5), 13801-13814.

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Western Blot Analysis of Chondrocyte Proteins

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Western blot analysis of whole-cell extracts was performed as previously described.³³ (link) The primary antibodies were anti-Aggrecan (1:1,000, MABT83; Millipore, USA), anti-Collagen II (1:1,000, ab188570; Abcam, USA), anti-SOX9 (1:1,000, AB5535; Millipore, USA), anti-Collagen X (1:1,000, ab182563; Abcam, USA), anti-ADAMTS4 (1:800, ab185722; Abcam, USA), anti-MMP13 (1:2,000, ab39012; Abcam, USA), anti-Smad2 (1:1,000, #5339; Cell Signaling Technology [CST], USA), anti-Smad3 (1:1,000, #9523; CST, USA), anti-phospho-Smad2 (1:1,000, ab53100; Abcam, USA), anti-phospho-Smad3 (1:1,000, ab52903; Abcam, USA), anti-Runx1 (1:800, ab23980; Abcam, USA), and anti-GAPDH (1:2,000, #97166; CST, USA). GAPDH was utilized as an internal control. The grayscale values of the blots were quantitated with ImageJ software by following the instructions. The grayscale value of each target protein was normalized to that of GAPDH.

Zhao X., Meng F., Hu S., Yang Z., Huang H., Pang R., Wen X., Kang Y, & Zhang Z. (2020). The Synovium Attenuates Cartilage Degeneration in KOA through Activation of the Smad2/3-Runx1 Cascade and Chondrogenesis-related miRNAs. Molecular Therapy. Nucleic Acids, 22, 832-845.

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Chondrogenic Differentiation of Mesenchymal Stem Cells

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Dulbecco’s modified Eagle medium (DMEM) and Penicillin-streptomycin from Welgene (Namcheon-myeon, Gyeongsangbuk-do, Korea). Fetal bovine serum (FBS) from Gibco (Carlsbad, CA, USA). Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), dexamethasone, and lithium chloride from Sigma-Aldrich (St. Louis, MO, USA). Recombinant Human IL-1β from PeproTech (Cranbury, NJ, USA). H89 (#9844) and Okadiac acid (#5934) from Cell Signaling Technology (Bervely, MA, USA). Parathyroid Hormone (7-34), Human Recombinant (228-11343-2) from RayBiotech (Peachtree Corners, GA, USA). Antibodies against VEGFA (ab46154, 1:1000) and Collagen X (ab182563, 1:1000) from Abcam (Cambridge, UK). RUNX2 (#12566, 1:1000), HDAC4 (#5392, 1:1000), SOX9 (#82630, 1:1000), β-catenin (#8480, 1:1000), Lamin A/C (#4777, 1:2000), HRP linked anti-mouse IgG (#7076, 1:5000), and HRP linked anti-rabbit IgG (#7074, 1:5000) from Cell Signaling Technology (Bervely, MA, USA). β-actin (A5411, 1:5000) from Sigma Aldrich (St. Louis, MO, USA). Antibody Collagen II (sc-52658, 1:1000) and PTH/PTHrP-R (sc-12722, 1:1000) from Santa Cruz Biotechnology (Dallas, TX, USA). HSP90 (13171-1-1AP, 1:2500) from Proteintech (Rosemont, IL, USA).

Ali A., Park Y., Lee J., An H.J., Jin J.S., Lee J.H., Chang J., Kim D.K., Goo B., Park Y.C., Leem K.H., Seong S, & Kim W. (2021). In Vitro Study of Licorice on IL-1β-Induced Chondrocytes and In Silico Approach for Osteoarthritis. Pharmaceuticals, 14(12), 1337.

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Protein Expression Analysis Workflow

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Cells were collected by cell scraping. RIPA lysis buffer rich in protease inhibitors (Beyotime, China) was applied to extracted the proteins. BCA protein quantification kit (Beyotime, China) was applied to detect the proteins concentrations following the instructions of the manufacturer. Proteins were separated by electrophoresis using SDS-PAGE (Beijing Biosynthetic Biotechnology, China) and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, United States). The PVDF membrane was put into 5% skimmed milk powder blocking solution (Solarbio, China), and sealed for 2 h at room temperature. The sealed PVDF membrane was incubated with the anti-OCN (1:1000; Abcam, ab133612), OPN (1:1000; Abcam, ab214050), RUNX2 (1:2000; Abcam, ab76956), Collagen X (1:1000; Abcam, ab182563), MAPK1 (1:5000; Abcam, ab265600), GAPDH (1:2500; Abcam, ab9485), MEF2A (1:1000; Abcam, ab109420) overnight at 4°C.According to the source of the primary antibody, the secondary antibody was diluted with TBST solution and the PVDF membrane was put into the corresponding secondary antibody. An ultra-sensitive ECL chemiluminescence kit was applied to expose the membrane. The chemiluminescence of the protein bands was quantified by ImageJ software.

Wu Z., Yuan J., Li J., Du Z., Yin M., Cheng X., Liu X, & Jia J. (2022). Hsa_circ_0008870 suppresses bone formation of growth plate through inhibition of miR-185-3p/ MAPK1 axis in idiopathic short stature. Frontiers in Bioengineering and Biotechnology, 10, 1022830.

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Protein Expression Analysis in Cartilage Cells

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The cells in the culture were lysed using the RIPA buffer (Pierce, Rockford, IL, USA) with a protease inhibitor cocktail (Pierce). Total protein was separated by SDS-PAGE electrophoresis using the Novex Nu PAGE system (Invitrogen) and transferred to 0.45-μm PVDF membranes. Membranes were blocked in TBS-T buffer containing 5% nonfat milk for 1 h and incubated overnight at 4 °C with the following primary antibodies: anti-Collagen X (1:1,000, Abcam, #ab182563), anti-DLX5 (1:1,000, Abcam, #ab109737), anti-RUNX2 (1:1,000, Abcam, #ab264077), anti-SOX9 (1:1,000, Abcam, #ab185966), anti-MMP13 (1:1,000, Abcam, #ab51072), and anti-β-ACTIN (1:1,000, Beyotime, China, #AF0003) was used as the internal control. Then the membranes were washed using TBST and hybridized with the horseradish peroxidase (HRP)-linked antibody goat anti-rabbit IgG (1:4,000, Beyotime) or goat anti-mouse IgG for 1 h. Signal detection was carried out with an ECL system (Amersham Pharmacia, Piscataway, NJ, USA).

Chen J., Chen F., Wu X., Bian H., Chen C., Zhang X., Hei R., X.i.a.o.t.o.n.g.Y.a.n.g., Yuan H., Wang Q., Lu Y., Qiao L, & Zheng Q. (2023). DLX5 promotes Col10a1 expression and chondrocyte hypertrophy and is involved in osteoarthritis progression. Genes & Diseases, 10(5), 2097-2108.

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Protein Expression Analysis in Cartilage

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Cells were lysed and western blotting performed as previously described [5] . The following antibodies were used: type I, II, and X type collagen (ab34710, ab182563, ab34712) were purchased from Abcam, aggrecan (AB10031) from Millipore, Sox9 (LS-C368527) from LSBio and β-actin (sc-47778), β-catenin (sc-7963), goat anti-mouse IgG-HRP and goat anti-rabbit IgG-HRP from Santa Cruz Biotechnology.

Allas L., Rochoux Q., Leclercq S., Boumédiene K, & Baugé C. (2020). Development of a simple osteoarthritis model useful to predict in vitro the anti-hypertrophic action of drugs. Laboratory investigation; a journal of technical methods and pathology, 100(1).

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