Samples were sectioned at a thickness of 5 µm. All the sections were incubated for 1 hour with primary antibody directed against IL1B (Cell Signaling Technology, #2022, 1:200), COL2A1 (Santa Cruz, sc-52658, 1:200), COL4A1 (Thermo Fisher Scientific, PA5-85634, 1:500), LGALS1 (Cell Signaling Technology, #5418, 1:200), MMP3 (Abcam, ab223666, 1:500) after blocking endogenous peroxidase using 3% hydrogen peroxide for 5 minutes at room temperature. After rinsing, the sections were incubated for 1 hour with biotinylated horseradish peroxidase-conjugated goat antirabbit IgG. Diaminobenzidine was used to develop peroxidase staining.
Col2a1
Manufactured by Santa Cruz Biotechnology
Sourced in United States
COL2A1 is a gene that encodes the alpha-1 chain of type II collagen, a major structural component of cartilage. The protein produced by this gene is essential for the normal development and function of cartilage.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 13, by Santa Cruz Biotechnology (4 mentions)
Cox 2, by BD (2 mentions)
Wogonin, by Extrasynthese (2 mentions)
Antibodies specific for p erk1 2, by Cell Signaling Technology (2 mentions)
Antibodies specific for β actin, by Santa Cruz Biotechnology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Collagenase, by Roche (2 mentions)
Pronase, by Roche (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Col4a1, by Thermo Fisher Scientific (1 mentions)
Il 1b, by Cell Signaling Technology (1 mentions)
Lgals1, by Cell Signaling Technology (1 mentions)
Monoclonal anti β actin, by Merck Group (1 mentions)
Molecular imager chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Clarity western ecl substrate kit, by Bio-Rad (1 mentions)
Vibra cell, by Sonics (1 mentions)
Calci clear rapid, by National Diagnostics (1 mentions)
11 protocols using col2a1
1
Immunohistochemical Analysis of Inflammatory Markers
2
Western Blot Analysis of Chondrocyte Proteins
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The total proteins were prepared from cultured chondrocytes obtained as in ‘Quantitative analysis of gene expression by qRT‐PCR’ by lysis in the suspension of the RIPA Lysis and Extraction Buffer (Thermo Scientific) using Sonics Vibra Cell™ (Sonics & Materials, Newtown, CT, USA). Total cellular proteins were quantitatively analysed using the bicinchoninic acid (BCA) total protein quantitation assays 38 . Equal proteins were separated by 8.0% SDS‐PAGE, blotted onto nitrocellulose membrane (Bio‐Rad, Hercules, CA, USA). The membranes were blocked by skim milk powder solution in Tris‐buffered saline and then incubated with diluted primary antibodies of CTNNB1 (AVIVA, San Diego, CA, USA) against β‐catenin, COL2A1 (Santa Cruz) against COL II by specifically binding to the C‐terminal epitope of a highly conserved motif between human and rabbit, and COL1A (COL‐1; Santa Cruz) against COL I, respectively, with the Monoclonal Anti‐β‐Actin (Sigma‐Aldrich) against the house‐keeping gene β‐actin used as an internal control. The binding proteins were detected with conjugated secondary antibody, goat anti‐rabbit IgG‐HRP (Santa Cruz) and visualized using Clarity™ Western ECL Substrate Kit (Bio‐Rad). The images were captured and analysed using the Image Lab (Beta 1) Version 3.0.1 Changelist 40296 software associated with Molecular Imager®, ChemiDoc™ XRS+ Imaging System (Bio‐Rad).
3
Histological Assessment of Cartilage Models
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Knee joints were fixed with 3.7% formaldehyde, decalcified with Calci-Clear Rapid (National Diagnostics, Atlanta, GA, USA), embedded in paraffin, and cut into 100-μm sections. For histological analysis, the sections were deparaffinized in xylene and serially rehydrated in ethanol. The sections were sequentially stained with MT, H&E, Alcian blue and safranin O staining accrording to conventional protocols. To evaluate the histological assessment of the acute defect model, we used the O’Driscoll scoring system62 (link) and as demonstrated in Table S3 ; to evaluate the histological assessment of the OA models, we used modified Mankin and OARSI score systems for cartilage regeneration following as demonstrated in Table S4 .63 (link), 64 For IHC, primary antibodies against ACAN, COL2A1, MMP13 (all from Santa Cruz Biotechnology, USA), and COX2 (BD Biosciences, USA) were used. All histological results were evaluated fairly by independent blind assessment. The numbers of positively stained cells were calculated using ImageJ 1.51f software (NIH).
4
Western Blot Analysis of Cellular Proteins
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Protein was extracted from ATDC5 cells using the Protein Extraction Solution (MDL) supplemented with protease inhibitor (MDL). A BCA protein analysis kit (MDL) was used to quantify protein concentration. Protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (0.22 μm; Millipore, Bedford, USA). After blocking non-specific interactions, the membranes were incubated overnight at 4 °C with primary antibody against β-actin (MDL), Aggrecan, Bax, Bcl-2, LC3 (Proteintech), and COL2A1(Santa Cruz). Next, the membranes were incubated for 1 h with corresponding HRP*Polyclonal Goat Anti-Rabbit IgG (H + L) (MDL) conjugated with horseradish peroxidase at room temperature. Finally, the proteins were stained and captured using Pierce ECL Western Blotting Substrate (Thermo Scientific) on the Bio-Rad ChemiDoc MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
5
Chondrocyte Protein Isolation and Western Blot Analysis
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For isolation of cell proteins, HC‐a chondrocytes were washed with cold PBS twice and treated with RIPA buffer (Solarbio Biotech, Beijing, China). Cell lysates containing an equal amount of proteins were separated by electrophoresis and then transferred onto PVDF membranes (Immunoblot, Bio‐Rad). The membranes were blocked in TBST containing 5% non‐fat milk (Difco™ Skim Milk, BD) for 1 h at room temperature. After blocking, the membranes were incubated with primary antibodies against COL2A1 (Santa Cruz, USA), MMP13 (Santa Cruz, USA), PON2 (Abcam, USA), BCL2 (CST, USA), BAX (CST, USA), Cytochrome C (Abcam, USA), GAPDH (CST, USA) at 4 °C overnight, and then incubated with horseradish peroxidase(HRP)‐conjugated secondary antibody (Santa Cruz, USA). The antibody binding was detected using an ECL Western Blotting Substrate (Solarbio Science & Technology) and visualized using a Molecular Imager ChemiDoc XRS System (Bio‐Rad).
6
Wogonin Modulates Inflammatory Markers in Chondrocytes
7
Chondrocyte Culture and Signaling Pathway Analysis
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Media and other reagents for cell culture were purchased from HyClone Laboratories (Logan, UT, USA) or from Life Technologies (Carlsbad, CA, USA). Pronase and Collagenase were from Roche Diagnostics (Indianapolis, IN, USA). Recombinant human IL-1β was from R&D Systems (St Paul, MN, USA). Antibodies specific for p-ERK1/2, ERK1/2, p-JNK, JNK, p-P38, P38, p-c-Fos, c-Fos, p-c-Jun, c-Jun, and Iκβα were purchased from Cell Signaling Technology (Beverly, MA). Antibodies specific for β-Actin, MMP-13, IL-6, iNOS, and COL2A1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-COX-2 antibody was from R&D Systems. Baicalein, Baicalin, Wogonin, Scutellarein and Apigenin were purchased from Extrasynthese (GenayCedex, France).
8
Chondrocyte Signaling Pathway Analysis
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Transfected cells or limbs of knock-in mice were lysed in ice-cold radio immunoprecipitation assay (RIPA) buffer containing 0.01% protease (Sigma-Aldrich, USA) for 30 min at 4°C. Cell lysis was centrifuged at 12000 rpm for 15 min at 4°C and the protein concentrations were quantified using the Bicinchoninic Acid (BCA) kit (Sigma-Aldrich, USA). SDS-PAGE and western blotting were performed using standard techniques. The primary antibodies of GDF5, SOX9, COL2A1, SMURF1, ID1 and phosphorylated SMAD1/5/8 (Santa Cruz, USA) were used at a dilution of 1:1000, and β-tubulin (Abcam, USA) was used at a dilution of 1:10000 in PBS-Tween 20.
9
Quantifying Epithelial-Mesenchymal Transition
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Extracts of total cellular proteins were prepared by scraping the cells into Mammalian Protein Extraction Reagent (Thermo Scientific Pierce, No. 78503, Shanghai, China). The protein samples were separated on the 12% SDS-PAGE and electrotransferred onto polyvinylidene difluoride-nitrocellulose membranes (Immobilon-P; Millipore Corporation, Bedford, MA, USA). The following primary antibodies were used: E-cadherin (SC-8426, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), Cytokeratin19 (CK19) (SC-56371, Santa Cruz Biotechnology, Inc.), Zeb1 (ABIN755113, Antibodies-online, Shanghai, China), Snail1 (ABIN182776, Antibodies-online, Shanghai, China), Sox9 (SC-20095, Santa Cruz Biotechnology, Inc.), Col2A1 (SC-52658, Santa Cruz Biotechnology, Inc.), Runx2 (No. 12556, Cell Signaling Technology, Boston, MA, USA), OPN (SC-21742, Santa Cruz Biotechnology, Inc.), STIM1 (SC-166541, Santa Cruz Biotechnology, Inc.), ORAI1 (SC-68895, Santa Cruz Biotechnology, Inc.). HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies (Boster, BA1051, Wuhan, China) were used and detected by electrochemiluminescence (Amersham Pharmacia Biotech, Aylesbury, UK).
10
Fabrication and Characterization of Alginate-Based Bone Tissue Scaffolds
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Human MG-63 cell line was provided by the Iranian National Cell Bank (Pasture Institute; Iran). Pen/Strep, FBS, PBS, DMEM/LG, and 0.25% Trypsin–EDTA solution were obtained from Gibco (UK). Sodium alginate (I-1G, high content of guluronic acid, and MW: 70 kDa) was purchased from Kimica (Tokyo, Japan). BCIP/NBT alkaline phosphates color development kit, lysozyme, nano-hydroxyapatite, p-NPP, barium chloride, strontium chloride, DMSO, and MTT powder were purchased from Sigma-Aldrich. Alizarin Red S, calcium chloride, magnesium chloride, Tris-Based, sodium chloride, paraformaldehyde, and trisodium citrate dehydrate were obtained from Wako Pure Chemical Corporation (Osaka, Japan). Collagen type I was obtained from SBPE Company (Tabriz, Iran). SOD, TAC, and Superoxide dismutase assay kits were purchased from Randox (Crumlin, United Kingdom). Sodium sulfate was obtained from Merck (Germany). RIPA lysis buffer kit, HRP-conjugated anti-IgG, B-actin, COL1A1, COL2A1, OCN, and SOX-9 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).
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