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.
Anti col2
Manufactured by Abcam
Sourced in United States
Anti-Col2 is a laboratory reagent used to detect and quantify the presence of type II collagen in biological samples. It functions as a specific antibody that binds to type II collagen, enabling researchers to analyze and measure this important structural protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti acan, by Abcam (2 mentions)
Anti mmp13, by Abcam (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Gapdh, by Keygen Biotech (1 mentions)
Goat anti rabbit igg hrp secondary antibody, by Abcam (1 mentions)
Mmp13, by Abcam (1 mentions)
Anti col1, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Phosphatase inhibitor cocktail 1, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Anti filamin a, by Abcam (1 mentions)
Anti rac1, by Proteintech (1 mentions)
Anti src, by Proteintech (1 mentions)
Anti n cdh, by Proteintech (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
5 protocols using anti col2
1
Immunohistochemical Analysis of Cartilage Markers
2
Hydrogel-Mediated Protein Expression Analysis
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WB was conducted to evaluate the protein expression level after culturing BMSCs and chondrocytes with hydrogels for 7days or 5 days. The protein of the cells was extracted with a Total ProteoExtract Kit (KeyGEN, Nanjing, China). After measuring the concentration with a BCA Protein Assay Kit (KeyGEN, Nanjing, China), the proteins were separated with SDS-PAGE electrophoresis and transferred to PVDF membranes. Blocking with 5% BSA for 1 h, the PVDF membranes were incubated with the primary antibodies at 4 °C. The antibodies applied include anti-SOX-9 (Proteinteck, China), anti-COL-2 (Abcam, UK), anti-RUNX2 (Proteinteck, China), anti-COL I (Proteinteck, China), GAPDH (KeyGEN, Nanjing, China). After washing in Tris buffered saline–Tween (TBST), the blots were developed with an HRP-conjugated secondary antibody and visualized with ChemiDoc MP Imaging System (Bio-rad). Protein band intensity was quantified using ImageJ and normalized to the corresponding GAPDH bands (NIH, USA).
3
Immunohistochemical Evaluation of Extracellular Matrix in Intervertebral Discs
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IHC staining (Boster Biological Technology, China) was performed according to the manufacturer's protocol to further evaluate the matrix proteoglycan complex deposition and expression in NP tissues. Briefly, the harvested IVD samples were fixed with 4% paraformaldehyde for 48 hours, and then were decalcified in 10% EDTA for 4 weeks. Afterwards, the treated samples were sequentially fixed with paraformaldehyde for 24 h, embedded in paraffin and sectioned at 4 mm. The sections were treated with 3% H2O2 for 15 min at room temperature to eliminate endogenous peroxidase activity and incubated with 0.12% trypsin for 30 min at 37 °C to retrieve the antigen, before being blocked with normal goat serum for 15 min at room temperature. Next, the sections were incubated with primary antibodies (anti-ACAN (1:100; Abcam, USA), and anti-Col2 (1:100; Abcam, USA)) overnight at 4°C and then incubated with goat anti-rabbit IgG-HRP secondary antibody. The stained sections were photographed under a microscope (Olympus, Japan). Finally, the sections were counterstained with Harris's haematoxylin and imaged under an optical microscope. The average optical density (AOD) of five randomly selected visual fields (per immunohistochemical slice) was measured using the ImageJ analysis system.
4
Histological Analysis of Rat Femurs
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The rats were sacrificed and whole femurs were collected after 8 weeks of treatment. Samples were fixed in 4% paraformaldehyde (pH 7.5) for 1 day and decalcified in decalcifying solution for 21 days, the samples were then embedded in paraffin and cut into 5 μm sections. Sagittal sections from the growth plate defect were stained with hematoxylin, and eosin (HE, saffron O/Fast Green (SO, ServiceBio), and immunohistochemical staining. The rabbit anti-COL-2 (Abcam), MMP-13 (Abcam), Arg-1 (Gentex), and iNOS (Gentex) primary antibodies (Table 2 ) were used for immunohistochemistry.
5
Western Blot Analysis of Cartilage Proteins
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The NPCs and NPC spheroids were collected and lysed in lysis buffer (Beyotime, China) containing a mixture of protease inhibitors phenylmethanesulfonyl fluoride (PMSF, Beyotime) and phosphatase inhibitor cock-tail I (Sigma, USA). A BCA protein quantification kit was used to determine the protein concentration of each sample according to the manufacturer's protocol. Depending on the results, equivalent amounts of protein (20 μg) were loaded on the 10% or 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. After electrophoresis was completed, the separation gel was removed, and the target protein in the gel was transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). After blocking with the 3% bovine serum albumin (BSA) blocking solution, the membranes were incubated with the primary and secondary antibodies, developed, fixed and exposed. The primary antibodies used in this study were as follows: anti-ACAN (1:1000; Abcam, USA), anti-Col1 (1:1000; Abcam, USA), anti-Col2 (1:1000; Abcam, USA), anti-matrix metallopeptidase-13 (MMP-13; 1:500; Proteintech, China); anti-N-CDH (1:500; Proteintech, China), anti-Integrinβ1 (ITGβ1; 1:1000; Abcam, USA), anti-Filamin A (1:1000; Abcam, USA), anti-Rac1 (1:500; Proteintech, China), anti-p-FAK 1:500; Proteintech, China), anti-Src (1:500; Proteintech, China), and anti-GAPDH (1:500; Proteintech, China).
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