Anti collagen 2

Manufactured by Abcam

Sourced in United States, United Kingdom

Anti-collagen II is a laboratory product used to detect and measure the presence of collagen type II in samples. It is an antibody-based tool that can be used for various research applications involving the analysis of collagen type II.

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Lab products found in correlation

Anti aggrecan, by Abcam (8 mentions) Anti mmp13, by Abcam (6 mentions) Anti collagen x, by Abcam (3 mentions) Anti adamts5, by Abcam (3 mentions) Anti sox9, by Abcam (3 mentions) Bca kit, by Thermo Fisher Scientific (3 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 mmp 3, by Abcam (2 mentions) Anti adamts4, by Abcam (2 mentions) Anti gapdh, by Abcam (2 mentions) Bovine serum albumin (bsa), by Merck Group (2 mentions) Anti cd24, by Abcam (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions) Pvdf membrane, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Image pro plus 6, by Media Cybernetics (2 mentions) Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)

Spelling variants of this product

Collagen II (57 citations) Anti-collagen II antibody (9 citations) Rabbit anti-collagen II (4 citations) Anti-type II collagen antibody (3 citations) Anti-collagen type II (3 citations) Mouse anti-collagen II (3 citations) Rabbit anti‐collagen II antibody (2 citations) Rabbit anti-human collagen II antibody (2 citations) Mouse monoclonal anti-collagen II (2 citations)

27 protocols using anti collagen 2

Western Blot Analysis of ECM Proteins

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After tissues were homogenized in liquid nitrogen, the homogenate was lysed on ice for 30 min in lysis buffer (BioTeKe, Beijing, China). The lysates (20–40 μg) of total protein were loaded per well and separated on a 10% SDS-polyacrylamide gel. Primary antibodies were anti-CTGF (1:5000 dilution, Abcam, Shanghai, China), anti-collagen-II (1:5000 dilution, Abcam), anti-collagen-IV (1:5000 dilution, Abcam), anti-Smad 2/3 (1:1000 dilution, Cell Signaling Technology, Boston, MA), anti-Smad 7 (1:1000 dilution, Santa Cruz Biotech, Santa Cruz, CA), anti-p-Smad2 (1:1000 dilution, Cell Signaling Technology), and anti-actin antibodies (1:5000 dilution, Santa Cruz Biotech). The secondary antibody was a peroxidase-coupled anti-goat IgG (GE Healthcare). The membrane was exposed to ECL Hyperfilm (GE Healthcare), and the film was developed. The bands were quantified by densitometry using ImageJ. Results were from triplicate experiments.

Zou T., Zhu M., Ma Y.C., Xiao F., Yu X., Xu L., Ma L.Q., Yang J, & Dong J.Z. (2018). MicroRNA-410-5p exacerbates high-fat diet-induced cardiac remodeling in mice in an endocrine fashion. Scientific Reports, 8, 8780.

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Immunohistochemical Analysis of Chondrocyte Markers

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The expression of collagen II, collagen X and PKC was detected using immunohistochemistry. The slices were dewaxed in xylene, dehydrated in graded ethanol and incubated in 3% H₂O₂ at 37°C for 10 minutes. Next, they were washed in PBS for 5 minutes thrice, boiled in 0.01 M citric acid buffer for antigen retrieval (95°C, 15‐20 minutes) and blocked in goat serum for 10 minutes at 37°C. Slices were then incubated with primary antibody (anti‐Collagen II (1:200), anti‐Collagen X (1:50), anti‐PKC‐ε (1:50) and anti‐PKC‐δ (1:1000), Abcam, USA) at 4°C overnight and with biotin‐labelled secondary antibody (Bioworld) for 30 minutes at 37°C. Slices were counterstained with haematoxylin and observed under a light microscope.

Ge J., Cheng X., Yan Q., Wu C., Wang Y., Yu H., Yang H., Zhou F, & Zou J. (2020). Calcitonin inhibits intervertebral disc degeneration by regulating protein kinase C. Journal of Cellular and Molecular Medicine, 24(15), 8650-8661.

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Chondrocyte Protein Extraction and Western Blot

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The reagent RIPA Lysis Buffer (Thermo Fisher Scientific) was used to extract the total protein of the cultivated chondrocyte or isolated cartilage tissue. RIPA was supplemented with protease inhibitor PMSF. Protein concentration was determined by a BCA kit (Thermo Fisher Scientific). Protein was mixed with protein loading buffer (Thermo Fisher Scientific) and loaded to a 12% SDS-PAGE gel for electrophoresis. Afterward, the gel was transferred to PVDF membrane (Thermo Fisher Scientific). After a blocking step with a blocking buffer, the membrane was incubated with primary monoclonal antibodies at 4 °C overnight. After washing, the membrane was incubated with secondary antibody. Finally, signal was detected by an ECL method. The primary antibodies used in this study includes anti-Aggrecan (1:1000, Abcam), anti-Collagen II (1:1000, Abcam), anti-β-actin (1:3000, Abcam), anti-ADAM8 (1:1000, Abcam), anti-p-ERK (1:1000, Abcam), anti-ERK (1:2000, Abcam), anti-p- NF-κB p65 (1:1000, Abcam), anti-NF-κB p65 (1:2000, Abcam), anti-MMP9 (1:1000, Abcam), anti-Notch1 (1:1000, Abcam), anti-Hes1 (1:2000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h at RT.

Duan B., Liu Y., Hu H., Shi F.G., Liu Y.L., Xue H., Yun X.Y., Yan M.Y., Han X.R., Chen A.F., Wang Y, & Li Z.H. (2019). Notch1-ADAM8 positive feed-back loop regulates the degradation of chondrogenic extracellular matrix and osteoarthritis progression. Cell Communication and Signaling : CCS, 17, 134.

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Investigating Molecular Pathways in Osteoarthritis

Cited 2 times

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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)].

Li Z., Huang Z., Zhang H., Lu J., Tian Y., Piao S., Lin Z, & Bai L. (2021). Moderate-intensity exercise alleviates pyroptosis by promoting autophagy in osteoarthritis via the P2X7/AMPK/mTOR axis. Cell Death Discovery, 7, 346.

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Collagen II Immunohistochemistry Protocol

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Primary NPCs were treated with different concentrations of IL-1β and/or Genistein for the indicated time. Cells were then fixed with 4% PFA for 15-20 min and 3 % H₂O₂ was added for 10 min. Cells were blocked with goat serum (Abcam, CA, USA) for 20 min, followed by incubation with the primary antibody (anti-collagen II, Abcam) at 4 °C overnight. The next day samples were incubated with HRP-conjugated second antibody (Bioworld, CA, USA) at 37 °C for 40 min. DAB was added and the staining was observed under a microscope. Where hematoxylin was used for nuclear staining, this was followed by dehydration with 1% hydrochloric acid in ethanol, a PBS wash, then sealing and microscope examination.

Ge J., Zhou Q., Cheng X., Qian J., Yan Q., Wu C., Chen Y., Yang H, & Zou J. (2020). The protein tyrosine kinase inhibitor, Genistein, delays intervertebral disc degeneration in rats by inhibiting the p38 pathway-mediated inflammatory response. Aging (Albany NY), 12(3), 2246-2260.

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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.

Zhou P., Xu P., Yu W, & Li H. (2022). MiR-206 improves intervertebral disk degeneration by targeting GJA1. Journal of Orthopaedic Surgery and Research, 17, 157.

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Histological Analysis of Cartilage Formation

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The retrieved implants were cut in half longitudinally and fixed with 4 % paraformaldehyde solution. The fixed samples were then embedded in paraffin wax and sectioned into 25 μm thickness slices with a microtome. After deparaffinization, the sliced samples were treated with 0.5 % Triton X-100 solution (Biosesang co. Korea) for permeabilization. 1 % (w/v) bovine serum albumin (Sigma Aldrich, USA) solution was used to reduce the non-specific background. Sections were immunostained with a primary antibody against anti-Collagen II (Abcam, UK), and anti-Aggrecan (Abcam, UK) to make the cartilaginous ECM formation visible, followed by incubation with Alexa Flour secondary antibody (Invitrogen, USA) following the manufacturer’s instructions. All samples were counterstained with DAPI (Vector Laboratories, USA). Stained samples were examined using a confocal microscope (LSM 8800, Zeiss, Germany).
For histological experiments, dewaxed sections were stained with staining kits for H&E (Abcam, UK), Alcian Blue (Abcam, UK), Safranin-O (ScienCell Research Laboratories co., USA), and Masson’s Trichrome (Abcam, UK) following the manufacturer’s instructions. Slides covered with cover-slips were scanned using an automatic digital slide scanner (Panoramic MIDI, 3DHISTECH, Hungary).

Hun Park J., Ahn M., Park S.H., Kim H., Bae M., Park W., Hollister S.J., Kim S.W, & Cho D.W. (2021). 3D bioprinting of a trachea-mimetic cellular construct of a clinically relevant size. Biomaterials, 279, 121246.

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Western Blot Analysis of Chondrocytes

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Control and treated chondrocytes were lysed, followed by SDS-PAGE electrophoresis as previously described [13 ]. The primary antibodies used were listed: anti-iNOS, anti-COX-2, anti-β-actin, and HRP-conjugated secondary antibody from Santa Cruz Biotechnology, Santa Cruz, CA; and anti-ADAMTS-4, anti-ADAMTS-5, anti-aggrecan, anti-collagen II, anti-p65, anti-p-p65, anti-p-IκBα, and anti-IκBα from Abcam. Finally, the bands were visualized with the ECL reagent.

Zhu W., Zhang Y., Li Y, & Wu H. (2022). Glaucocalyxin A Attenuates IL-1β-Induced Inflammatory Response and Cartilage Degradation in Osteoarthritis Chondrocytes via Inhibiting the Activation of NF-κB Signaling Pathway. Disease Markers, 2022, 6516246.

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Protein Expression Analysis of Extracellular Matrix

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Cells were lysed and protein was extracted using RIPA buffer (Invitrogen). In addition, the BCA protein assay kit (Beyotime Bio, Shanghai, China) was used to detect the concentration of protein. Equal amounts of protein (30 μg) samples were separated using 10% SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% skim milk, the membranes and the following primary antibodies were incubated overnight at 4°C: anti-HGF (EPR12230, 1:1000, Abcam, Cambridge, MA, USA), anti-MMP-3 (EP1186Y, 1:1000, Abcam), anti-MMP-13 (1: 2000; Abcam), anti-Aggrecan (6-B-4, 1:1000, Abcam), anti-Collagen II (EPR12268, 1:1000, Abcam), anti-c-MET (EPR19067, 1:1000, Abcam), anti-p-c-MET (EP2367Y, 1:1000, Abcam), and GAPDH (6C5, 1:2000, Abcam). GAPDH protein was used as the inner control. After washing, the membranes were incubated with the HRP-conjugated secondary antibody at 1:2000 dilution for 1 h at room temperature. The resulting bands were detected with an ECL detection system (Millipore).

Que W., Liu H, & Yang Q. (2022). CircPRKCH modulates extracellular matrix formation and metabolism by regulating the miR-145/HGF axis in osteoarthritis. Arthritis Research & Therapy, 24, 216.

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Cartilage Protein Extraction and Analysis

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Total proteins were extracted from chondrocytes or cartilage tissues with RIPA lysis buffer (CWBIO, China), and the concentration was measured using the BCA protein assay (Beyotime, China). Then, proteins were electrophoresed on SDS-polyacrylamide gel and transferred to nitrocellulose membranes (Millipore, Carrigtwohill, Ireland). After blocking with nonfat milk, the membranes were incubated with a primary antibody overnight at 4°C, washed, and then incubated with a secondary antibody for 1 h each at room temperature. The antibodies used were anti-collagen II (dilution 1 : 5,000; Abcam, UK), anti-MMP-13 (dilution 1 : 3,000; Abcam, UK), GAPDH (dilution 1 : 1,000; Abcam, UK), and goat anti-rabbit secondary antibody (dilution 1 : 5,000; Abcam, UK). The results were quantified, and the images were processed using ImageJ software. GAPDH was used as an internal loading control.

Apizi X., Talifujiang D., Kasimu A., Zhang X., Yiming A., Ma X., Song Q, & Wang D. (2021). Circular RNA mmu_circ_0001598 Contributes to IL-1β-Induced Osteoarthritis Progression by Regulating miR-127-3p. Journal of Healthcare Engineering, 2021, 2793379.

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