Immunofluorescence was performed as previously described [28 (link),29 (link)]. Briefly, the bone sections were incubated with individual primary antibodies against mouse CD31 (ab28364; Abcam), endomucin (V.7C7; Santa Cruz), Ki67 (AF7617; R&D), beta-catenin (8480, CST), osterix (bs-1110R; Bioss), osteocalcin (bs-0470R; Bioss), Runx2 (bs-1134R; Bioss), DLL4 (bs-6044R; Bioss), Notch1 (bs-1335R; Bioss), Noggin (bs-2975R; Bioss), CXCR4 D1S7W; Cell Signaling Technology), integrin αvβ3 (bs-1310R; Bioss), CD90 (bs-20640R; Bioss), CD105 (bs-0579R; Bioss), CD271 (bs-0161R; Bioss), OPN (bs-23258R; Bioss), and phospho-VEGFR2(bs-2674R; Bioss) overnight at 4 °C. Subsequently, the samples were incubated with fluorescence-coupled secondary antibodies for 1 h at 37 °C in the dark. Images were collected using a confocal laser microscope (LSM 780; Carl Zeiss).
Runx2
Manufactured by Bioss Antibodies
Sourced in United States
Runx2 is a protein that plays a key role in the regulation of bone and cartilage development. It is a transcription factor that activates the expression of genes involved in the differentiation of osteoblasts, the cells responsible for bone formation. Runx2 is essential for the initial stages of bone development and the maintenance of mature bone tissue.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780, by Zeiss (2 mentions)
Osteocalcin, by Bioss Antibodies (1 mentions)
Bs 1134r, by Bioss Antibodies (1 mentions)
Bs 1310r, by Bioss Antibodies (1 mentions)
Ab28364, by Abcam (1 mentions)
Endomucin v 7c7, by Santa Cruz Biotechnology (1 mentions)
Bs 1110r, by Bioss Antibodies (1 mentions)
Bs 0470r, by Bioss Antibodies (1 mentions)
Cd105, by Bioss Antibodies (1 mentions)
Bs 0579r, by Bioss Antibodies (1 mentions)
Bs 23258r, by Bioss Antibodies (1 mentions)
Cxcr4 d1s7w, by Cell Signaling Technology (1 mentions)
Phospho vegfr2 bs 2674r, by Bioss Antibodies (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
α modified minimum essential medium α mem, by Thermo Fisher Scientific (1 mentions)
Bmp 2, by Bioss Antibodies (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by MP Biomedicals (1 mentions)
4 protocols using runx2
1
Immunofluorescence Analysis of Bone Markers
2
Chondroitin Sulfate Promotes Osteogenesis
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CS was supplied by Zhengzhou Corey Fine Chemical (MW 30–36 kDa) (with 55% deacetylation), TGF-β3 and RHC were provided by Jinan University Biopharmaceutical R and D Center (Guangzhou, China), and α-modified minimum essential medium (α-MEM), fetal bovine serum (FBS), and trypsin-EDTA were purchased from Gibco BRL. Penicillin and streptomycin (P/S) were purchased from MD Bio, China. MTT was purchased from MP Biomedicals (United States). Antibodies against collagen-1 (COL Ⅰ) were purchased from Affinity Biosciences (Cincinnati, OH, United States), BMP-2 and RUNX2 were purchased from Bioss (Boston, MA, United States), and GAPDH and an horseradish peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology (Boston, MA, United States).
3
Bone Protein Quantification and Analysis
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The proteins of animal bone tissue and osteoblasts were quantified using a BCA protein assay kit and separated by electrophoresis in 10% sodium dodecyl sulphate-polyacrylamide gel before being transferred to PVDF membranes (Millipore, United States). PVDF membrane was combined with antibodies of β-actin, ERK1/2 (Proteintech, United States), Smad4 (Proteintech, United States), Runx2 (Bioss, Beijing, China), incubating overnight at 4°C, then shaken with secondary antibody at room temperature for 1 h. Exposure was taken in a TANON gel imager.
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted from bone using RIPA buffer (Beyotime Institute of Biotechnology, Haimen, China) containing 1% phenylmethylsulphonyl fluoride. A bicinchoninic acid (BCA) assay kit (Beyotime Institute of Biotechnology) was used to determine the protein concentration. The sample was resolved using 10% SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and blocked with 5% skim milk. The membrane was incubated with primary antibodies against Zip1 (1: 100; Santa Cruz Biotechnology, Santa Cruz, California, USA), NPY (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA), NPY1R (1:500; Abcam), type I collagen α1 chain (COL1A1; 1:300; CST, Boston, MA, USA), runt-related transcription factor 2 (Runx2; 1:1000; Bioss, Beijing, China) and GAPDH (1:1000; Key-GEN, Nanjing, China) overnight at 4°C. Membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibodies, and the proteins were visualized using enhanced chemiluminescence (Beyotime Institute of Biotechnology). The expression of target genes was evaluated using gray value analysis, which was normalized to the GAPDH reference gene.
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