Immunohistochemical staining was performed as previously described [44 (link)]. Tissue slices were deparaffinized and rehydrated. After that, microwave was used to retrieve the antigen. Next, we used 3% hydrogen peroxide to block the endogenous peroxidase activity and 5% BSA to block the non-specific binding sites under room temperature. The primary antibodies were incubated with slices overnight at 4 °C. The next day, an appropriate HRP-conjugated secondary antibody was used to incubate with sections and counterstained with hematoxylin. Immunohistochemistry was done using the following primary antibodies: NR2F2 (A10251; Abclonal), EGR1 (55,117–1-AP; Proteintech), SOX5 (A6985; Abclonal), COMP (A13963; Abclonal), Collagen X (ab49945; Abcam), POSTN (A14556; Abclonal), CD68(A13286; Abclonal), and CD31(ab9498; Abcam).
Collagen x
Manufactured by Abcam
Sourced in United States
Collagen X is a type X collagen that is a component of the extracellular matrix in cartilage and other connective tissues. It plays a role in the regulation of cartilage and bone development.
Automatically generated - may contain errors
Lab products found in correlation
Collagen 2, by Chondrex (3 mentions)
3 3 diaminobenzidine tetrahydrochloride kit, by ZSGB-BIO (2 mentions)
Runx2, by Abcam (2 mentions)
Collagen 2, by Abcam (2 mentions)
Mmp13, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab9498, by Abcam (1 mentions)
Collagen 2, by Merck Group (1 mentions)
Activated caspase 3, by Bioworld Technology (1 mentions)
Dab horseradish peroxidase color development kit, by ZSGB-BIO (1 mentions)
Aggrecan, by Abcam (1 mentions)
Image pro plus 5, by Media Cybernetics (1 mentions)
Cleaved caspase 3, by Boster Bio (1 mentions)
Adamts5, by Abcam (1 mentions)
Collagen 2, by Novus Biologicals (1 mentions)
Gapdh, by Novus Biologicals (1 mentions)
Pparγ, by Santa Cruz Biotechnology (1 mentions)
Aggrecan, by Santa Cruz Biotechnology (1 mentions)
Lysis buffer, by Bio-Rad (1 mentions)
Enhanced chemiluminescence detection system, by Thermo Fisher Scientific (1 mentions)
12 protocols using collagen x
1
Immunohistochemistry for Protein Analysis
2
Assessing Cartilage Development in Growth Plates
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Limbs were fixed in 4% paraformaldehyde for 24 h and decalcified in 10% EDTA for 3 days. Paraffin sections (4-µm) were obtained and stained with Hematoxylin and Eosin (H&E), safranin O and toluidine blue. Mean values of heights of the reserve, proliferative, hypertrophic zones, and the total growth plate were calculated from measurements taken at 3 positions across the proximal tibia growth plates using ImageJ version 1.44p software. IHC/Immunocytochemistry (ICC) was performed with Hsitostain-Plus kit (ZSGB-BIO, China). Primary antibodies included: collagen II (Sigma, USA), sox 9 (Abcam, UK), collagen X (Abcam, UK), and activated-caspase 3 (Bioworld, USA). Detection was conducted with a DAB horseradish peroxidase color development kit (ZSGB-BIO, China). Semi quantitative analysis of IHC images through integrated optical density (IOD) was taken using Image-Pro Plus version 6.0.0.260 software.
3
Immunohistochemical Analysis of Bone Markers
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Decalcified bone sections were deparaffinized with xylene, and endogenous peroxidase activity was quenched with 3% H2O2 for 15 min., followed by antigen retrieval with trypsinization for 15 min. Then, sections were blocked with normal goat serum for 30 min. and incubated at 4°C overnight with primary antibody followed by the biotinylated secondary antibody and horseradish peroxidase‐conjugated streptavidin–biotin staining. Immunoreactivity was visualized with a 3, 3′‐diaminobenzidine tetrahydrochloride kit (ZSGB‐BIO, Beijing, China) followed by counterstaining with methyl green. Primary antibodies against the following proteins were used: collagen II (1:400; Chondrex, Redmond, WA, USA), collagen X (1:200; Abcam, Cambridge, MA, USA), Aggrecan (1:200 Abcam), MMP13 (1:200; PeproTech, Chicago, IL, USA), ADAMTS5 (1:200; Abcam), cleaved caspase‐3 (1:100; Boster, China) and phosphor‐P65 (1:200; CST, Danvers, MA, USA). The number of immunoreactive cells in sections was counted using Image‐Pro Plus 5.1 (Media Cybernetics, Rockville, MD, USA).
4
Hedgehog Signaling and Cartilage Proteins
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Expression of hedgehog signalling molecules and cartilage-related proteins was determined by western blotting as described previously [14 (link), 23 (link)]. Antibodies recognising Ptc (Aviva Systems Biology, San Diego, CA, USA; 1:500), Smo (Aviva Systems Biology; 1:1,600), Gli1 (Biorbyt, Cambridgeshire, UK; 1:1,000), Sox9 (OriGene, Rockville, MD, USA; 1:500), collagen II (Novus Biologicals, Littleton, CO, USA; 1:200), ACAN (Novus Biologicals; 1:100), collagen X (Abcam; 1:500), RUNX2 (Abcam; 1:500), ALP (Abcam; 1:500), PPAR-γ (Santa Cruz Biotechnology; 1:1000) and GAPDH (Novus Biologicals; 1:2000) were used as primary antibodies. The secondary antibody (1:2000) was purchased from Thermo (Waltham, MA, USA). GAPDH was used as an internal control protein.
5
Protein Expression Analysis of Chondrocytes
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The cells were washed three times with PBS and suspended in ice-cold lysis buffer (Bio-Rad, Hercules, CA, USA). The lysates were separated by 12% SDS-PAGE, and the proteins of equal quantity were transferred to nitrocellulose membranes. Membranes were blocked with Tris-buffered saline with Tween-20 buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% nonfat milk (Yili, China) and incubated overnight at 4°C with collagen II, collagen X (Abcam, Cambridge, MA, USA), aggrecan, Bcl-2, Bax, caspase-3 (Santa Cruz, Dallas, TX, USA), p21, p27, CDK2, CDK4 (Cell Signaling Technology, Danvers, MA, USA), p-ERK1/2, and ERK1/2 (Abcam, Cambridge, MA, USA) primary antibodies. The following day, the membranes were washed and incubated with the corresponding secondary antibodies at room temperature (22-28°C) for 1 h. An enhanced chemiluminescence detection system (Thermo Scientific, MA, USA) was finally used to determine the emission of the membrane. The experiments were performed in three independent replicates. The GAPDH expression level was used as an internal control. The Western blot results were quantified with an image analyzer (Quantity One-4,2,0, Bio-Rad, Hercules, CA) and were normalized to GAPDH immunostaining. The relative protein expression was compared with the control group.
6
Telomerase-Transduced Mouse Mesenchymal Stem Cell Protocol
7
Immunohistochemical Analysis of Cartilage
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Decalcified bone sections were deparaffinized with xylene, and endogenous peroxidase activity was quenched by treatment with 3% H2O2 for 15 min, followed by antigen retrieval by trypsinization for 10 min. Sections were then blocked with normal goat serum for 30 min and incubated at 4 °C overnight with primary antibody followed by the appropriate biotinylated secondary antibody and horseradish peroxidase-conjugated streptavidin-biotin staining. Immunoreactivity was visualized with a 3,3′-diaminobenzidine tetrahydrochloride kit (ZSGB-BIO, Beijing, China) followed by counterstaining with Methyl green. Primary antibodies against the following proteins were used: collagen II (1:400; Chondrex, Redmond, WA, USA), FGFR3 (1:200; Santa Cruz Biotechnology, Dallas, Texas, USA), Osteocalcin (1:200; Santa Cruz Biotechnology), PCNA (1:200; Epitomics, Burlingame, CA, USA), Collagen X (1:200; Abcam, Cambridge, MA, USA), Aggrecan (1:200; Millipore, Billerica, MA, USA), MMP13 (1:200; Proteintech, Chicago, IL, USA), ADANTS5(1:200; Abcam), lubricin (1:600; Abcam), IHH (1:100; Abcam) and RUNX2 (1:200; Santa Cruz Biotechnology). The number of immunoreactive cells in three central regions of condylar cartilage section was counted by using Image-Pro Plus 5.1.
8
Immunohistochemical Analysis of Cartilage Markers
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The expression of collagen II (Abcam, Cambridge, MA, United States), collagen X (Abcam, Cambridge, MA, United States), and Smad3 (Cell Signaling Technology, Danvers, MA, United States) was detected using immunohistochemistry. The slides were dewaxed, dehydrated, and incubated in 3% H2O2 at 37°C for 10 min. The slides were then washed and boiled in 0.01M citric acid buffer for 20 min. Next, they were blocked in goat serum at 37°C for 10 min. Slides were then incubated with primary antibody at 4°C overnight and then in secondary antibody (Bioworld, Dublin, OH, United States) at 37°C for 30 min. Slides were finally counterstained with hematoxylin and subsequently evaluated with Image Pro Plus 6.0.
9
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.
10
Osteoarthritis Chondrocyte Protein Analysis
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Protein in tissues (sham and OA) and cells (different groups) were extracted using RIPA (Solarbio). Protein concentrations were determined by the BCA protein assay kit. Equal amounts of protein were taken from each sample, and transferring was performed with 10% SDS-PAGE and then transferred onto nitrocellulose filter (Millipore, USA), and probed with GAPDH (1:5000, Cell Signaling), Bax (1:1000, Cell Signaling), Bcl-2 (1:1000, Cell Signaling), cleaved caspase3 (1:1000, Cell Signaling), CRLF1 (1:1000, Abcam), Sox9 (1:1000, Abcam), ACAN (1:1000, Abcam), Col2a1 (1:1000, Abcam), MMP13 (1:1000, Abcam) and collagen X (1:1000, Abcam) antibody followed by appropriate secondary antibody. Proteins were detected by chemiluminescence (Millipore).
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