Sections were deparaffinized and hydrated and then placed in a citric acid solution to undergo 16 hours of incubation in a 60°C water bath for antigen retrieval, followed by incubation in a hydrogen peroxide solution for 10 minutes and blocking in 10% goat serum at 37°C for 30 minutes. The sections were then incubated in antibodies against p65, ADAMTS‐4 (purchased from ABclonal, Wuhan, China), Col1a2, MMP‐13, HIF‐1α (purchased from Abcam, Cambridge, MA, USA), and Col2a1 (purchased from Millipore, Burlington, MA, USA) at 4°C overnight, followed by incubation in secondary antibody (purchased from ABclonal, Wuhan, China) for 1 hour at room temperature, DAB chromogenesis, hematoxylin staining, and mounting to observe the expression of the corresponding proteins under a microscope.
Col2a1
Manufactured by Merck Group
Sourced in United States
COL2A1 is a laboratory equipment product. It is used to measure the expression levels of the COL2A1 gene, which encodes the type II collagen protein. The core function of COL2A1 is to provide quantitative data on the expression of this gene, which is important for research related to cartilage and bone development.
Automatically generated - may contain errors
Lab products found in correlation
Mmp13, by Abcam (4 mentions)
Aggrecan, by Abcam (2 mentions)
Adamts 4, by ABclonal (1 mentions)
Hif 1α, by Abcam (1 mentions)
Col1a2, by Abcam (1 mentions)
Mmp 13, by ABclonal (1 mentions)
Ferritin, by Abcam (1 mentions)
β catenin, by Abcam (1 mentions)
Collagen x, by Abcam (1 mentions)
Rabbit igg control antibody, by Merck Group (1 mentions)
Citric acid buffer, by Merck Group (1 mentions)
Mmp 13, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase labelled secondary antibody, by Cell Signaling Technology (1 mentions)
Interleukin 6 (il 6), by Merck Group (1 mentions)
Igf 1, by Merck Group (1 mentions)
Aggrecan, by Merck Group (1 mentions)
Caspase 1, by Merck Group (1 mentions)
9 protocols using col2a1
1
Immunohistochemical Profiling of Cartilage Markers
2
Histological Analysis of Intervertebral Disc Degeneration
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IVD specimens were fixed, decalcified, dehydrated, paraffin-embedded, and processed into serial 4 μm coronal sections. After deparaffinization and hydration, hematoxylin and eosin (H&E), Safranin O/Fast Green (SO/FG) and Masson staining were performed. For immunohistochemistry (IHC), slices were placed in citric acid solution (50mM) and incubated in a water bath at 60°C for 16 h to extract antigen, then incubated in 30% hydrogen peroxide solution for 10 minutes and blocked with 10% goat serum at 37°C for 1 hour. The sections were incubated in antibodies against MMP-13, OCN, NCOA4, LGR5 (purchased from ABclonal), Collagen X, p53, p16IKN4A, Ferritin, β-catenin (purchased from Abcam), and Col2a1 (purchased from Millipore) at 4°C overnight, followed by incubation in secondary antibody (purchased from Abcam) for 1 hour at room temperature, DAB chromogenesis and hematoxylin staining. For IF staining, the sections were incubated with a secondary antibody (purchased from Abcam) followed by staining with DAPI. In the quantitative analysis, the CEP height was calculated by using the mean value of the CEP height at 25%, 50%, and 75% of the coronal position of the IVD. The degree of IVDD was scored according to the modified scoring method in the previous literature.47 (link) IHC and IF staining were performed to compare protein expression by calculating the positive cell ratio.
3
Heat-Mediated Antigen Retrieval for Immunohistochemistry
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Heat‐mediated antigen retrieval was performed on paraffin‐embedded sections using citric acid buffer (Sigma‐Aldrich) warmed in a water bath at 100°C for 20 min. MMP‐13 (Abcam) antibody, COL2A1 (Sigma‐Aldrich) antibody and rabbit IgG control antibody (Sigma‐Aldrich) were used at 1:200 dilution. Quantification of positive cells/total cells was counted by two blinded operators.
4
Chondrocyte Protein Expression Analysis
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The chondrocytes and knee articular cartilage tissue were flushed three times with PBS and lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) for 30 min. The samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked with 5% skim-milk powder for 30 min at room temperature and then incubated with the corresponding primary antibodies of NLRP3, ASC, caspase-1, IL-6, IL-18, TNF-α, MMP13, ADAMTS4/5, IGF-1, aggrecan and Col2a1 (1:1000, Sigma-Aldrich, St. Louis, MO) at 4 °C overnight. After washing with PBS, the membranes were incubated with horseradish peroxidase-labelled secondary antibody (1:2000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature. Ultimately, the intensity of protein expression was measured by an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Carlsbad, CA). GAPDH served as control.
5
Western Blot Analysis of Autophagy and SIRT1 Markers
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Cells were homogenised in lysis buffer (RIPA buffer; Sigma-Aldrich), Ethylenediaminetetraacetic acid free protease inhibitor (Roche Pharmaceuticals), Phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich) and protein levels quantified by bicinchoninic acid assay (Thermo Fisher Scientific). For western blot analysis samples were probed overnight with primary antibody: Human SIRT1 (#9475; 1:1000; Cell Signalling Technology, MA, U.S.A), Mouse SIRT1 (07–131; 1:1000; Millipore, Massachusetts, U.S.A) or Beta-actin (β-actin) (A2228; 1:20000; Sigma-Aldrich), COL2A1 (SAB4500366; 1:1000; Sigma-Aldrich), SOX-9 (ab26414; 1:1000; Abcam, Cambridge, U.K.), ULK1 (NBP2-24738; 1:1000; Novus Biologicals, Cambridge, UK), Beclin1 (NB500-249; 1:1000; Novus Biologicals), LC3 (NB100-2220; 1:1000; Novus Biologicals), ATG5 (#12994; 1:1000; Cell Signalling Technology); ATG7 (#8558; 1:1000; Cell Signalling Technology), Acetyl-Lysine Antibody (#9441; 1:1000; Cell Signalling Technology), P62/SQSTM1 (NBP1-48320; 1:1000; Novus Biologicals). Secondary horseradish peroxidase (HRP)-conjugated anti-rabbit (#7074; 1:5000; Cell Signalling Technology), Secondary HRP-conjugated anti-mouse (#7076; 1:5000; Cell Signalling Technology)
6
Immunostaining of Cartilage Degeneration
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Tissue samples were evaluated for indirect immunostaining with the mouse monoclonal antibodies Col2A1 (M2139) and MMP-13 (C-3) from Santa Cruz Biotechnology, using a biotinylated anti-mouse secondary antibody (Donkey anti-mouse IgG (H + L) Biotin, Millipore, Burlington, MA, USA) and the avidin–biotin complex (ABC Elite PK-6100, Vector, Burlingame, CA, USA). Antibody binding was revealed with the DAB-peroxidase kit (Peroxidase Substrate Kit DAB SK-4100 Vector) and counterstaining with Crystal Violet (C5042, Sigma, St. Louis, MO, USA).
The presence of type II collagen (Col2A1) is related to the presence of healthy tissue, which, when hypertrophied after damage, begins to be replaced by type X collagen. On the other hand, metalloproteinase 13 (MMP- 13) is the most widely used marker of inflammation in the evaluation of cartilage, as it is the main protease in articular cartilage degradation [21 ,22 (link)].
The presence of type II collagen (Col2A1) is related to the presence of healthy tissue, which, when hypertrophied after damage, begins to be replaced by type X collagen. On the other hand, metalloproteinase 13 (MMP- 13) is the most widely used marker of inflammation in the evaluation of cartilage, as it is the main protease in articular cartilage degradation [21 ,22 (link)].
7
Quantification of Extracellular Matrix Proteins
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The total proteins in cells were extracted and the protein concentrations were determined by BCA protein assay kit (Beyotime). 30 μg of the total proteins were applied to 10% SDS-PAGE and then transferred onto PVDF membranes. After being blocked in TBS containing 5% non-fat milk for 1 h, the membranes were co-incubated with the primary antibodies at 4°C overnight against MMP-13 (1:1,000 dilutions, Cell signaling technology, Cat. no.69926), Col2a1 (1:1,000 dilutions, Sigma, Cat. no.SAB4500366), and GAPDH (1:1,000 dilutions, Cell signaling technology, Cat. no.5174), and then with the secondary antibody conjugated with peroxidase (1:2000 dilutions, Sigma-Aldrich, Cat. no. AP510). Protein bands were detected using the enhanced chemiluminescence detection system.
8
Immunohistochemical Analysis of Knee Cartilage
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Human knee joint sections obtained as described above were incubated with primary anti-OSCAR antibodies (Cat# SC34235, Santa Cruz Biotechnology, Inc., Dallas, TX, USA and Biorbyt, LLC, St Louis, USA, 1:200 dilution) at 4 °C overnight, followed by use of a 3,3′-diaminobenzidine peroxidase (DAB; horseradish peroxidase) substrate detection kit (Vector Laboratories, Inc., Burlingame, CA, USA) and counterstaining with hematoxylin. Immunohistochemistry was also conducted with antibodies against MMP3 (Cat# Ab53015; Abcam, Cambridge, MA, USA; 1:50 dilution), MMP13 (Cat# Ab51072; Abcam; 1:25 dilution), Aggrecan (Cat# Ab3778; Abcam; 1:100 dilution), COL2A1 (Cat# MAB8887; Sigma-Aldrich, St Louis, MO, USA; 1:50 dilution), ADAMTS5 (Cat# GTX100332; Genetex, Irvine, CA, USA; 1:200 dilution), SOX9 (Cat# ab185230; Abcam; 1:50 dilution), and PPARγ (Cat# ab59256; Abcam; 1:25 dilution).
9
Histological Analysis of Osteoarthritic Cartilage
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The knee joints were fixed in 10% formaldehyde at 4 °C for >48 h, decalcified in 0.5 M EDTA (pH 7.4) for 14 days, embedded in paraffin, cut into 5-μm sections, and stained with safranin-O with fast green counterstaining. Articular cartilage destruction was scored using OARSI. The tartrate-resistant acid phosphatase (TRAP)-staining kit was from Fujifilm Wako Pure Chemical Corporation (Japan). For immunohistochemistry, knee joint sections were incubated overnight at 4 °C with antibodies against MMP3 (Cat# Ab53015, 1:50), MMP13 (Cat# Ab39012, 1:50), Aggrecan (Cat# Ab1031, 1:100), pSmad3 (Cat# Ab52903, 1:100), Osterix (Cat# Ab22552, 1:400) (all from Abcam), or COL2A1 (Cat# MAB8887, 1:100; Sigma-Aldrich). Immunoactivity was detected with a DAB peroxidase-substrate detection kit (Vector Laboratories, USA). Nuclei were counterstained with hematoxylin. Samples were measured using OsteoMeasureXP (OsteoMetrics, Inc., Atlanta, GA, USA), Adobe photoshop (v19.1.3), and an Olympus DP72 charge-coupled device camera (v2.1, Olympus Corporation, Tokyo, Japan).
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