Sections of jaw and femur tissues were subjected to antigen retrieval (pH 6.0, Abcam, Tokyo, Japan), followed by peroxidase blocking (DAKO, Santa Clara, CA, USA). Anti-ALP (1:100, a gift from Hokkaido University, Hokkaido, Japan), anti-Runx2 (1:125, Abcam, Tokyo, Japan), anti-OSX (1:75, Abcam, Tokyo, Japan), anti-Mef2c (1:500, Abcam, Tokyo, Japan), anti-SOST (1:50, Abcam, Tokyo, Japan), anti-IL-1β (1:100, Abcam, Tokyo, Japan) and anti–HIF–1α (1:100, Abcam, Tokyo, Japan) were used as primary antibodies. The specimens were exposed to the same DAB reaction conditions, and all images were captured using a microscope (OLYMPUS, Tokyo, Japan).
Anti sost
Manufactured by Abcam
Sourced in Japan, United Kingdom, China, Italy, United States
Anti-SOST is a laboratory reagent used for detecting and quantifying the SOST protein in biological samples. SOST is a secreted glycoprotein that acts as a negative regulator of bone formation. Anti-SOST can be used in various research applications involving the study of bone metabolism and related diseases.
Automatically generated - may contain errors
Lab products found in correlation
Anti runx2, by Abcam (2 mentions)
Anti ocn, by Abcam (2 mentions)
Anti opn, by Merck Group (2 mentions)
Anti osx, by Abcam (1 mentions)
Peroxidase blocking, by Agilent Technologies (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti mef2c, by Abcam (1 mentions)
Anti alp, by Abcam (1 mentions)
Anti hif 1α, by Abcam (1 mentions)
Anti dkk1, by Abcam (1 mentions)
Anti gsk3β, by Abcam (1 mentions)
Anti akt, by Abcam (1 mentions)
Anti β catenin, by Abcam (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti pge2, by Abcam (1 mentions)
Anti pi3k, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Merck Group (1 mentions)
Enhanced chemiluminescent detection reagent, by Merck Group (1 mentions)
Proteasome inhibitor, by Beyotime (1 mentions)
5 protocols using anti sost
1
Immunohistochemical Analysis of Bone Markers
2
Protein Extraction and Western Blotting
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Whole cells were lysed using RIPA buffer (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.5% sodium dodecyl sulfate, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 1 μg/mL of pepstatin, and 1 μg/mL of leupeptin). Secretory protein was extracted by precipitation with trichloroacetic acid. Western blot analysis was performed with anti-PGE-2, anti-AKT, anti-PI3K, anti-GSK-3β, anti-β-catenin, anti-DKK-1, anti-SOST, or anti-β-actin antibodies (all from Abcam, Cambridge, UK).
3
Protein Expression Analysis of Osteogenic Markers
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Cells were lysed and the total cell protein was extracted in RIPA buffer containing a proteasome inhibitor (Beyotime, China). Protein concentrations were measured using a BCA protein assay kit (Beyotime). Equal protein amounts were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Shanghai, China). After blocking in 5% defatted milk for 2 h, the membranes were incubated with specific primary antibodies overnight at 4°C. Primary antibodies used in the present study include anti-GAPDH (1:10000, Proteintech, United States), anti-POSTN (1 μg/ml; Abcam, China), anti-SOST (1 μg/ml; Abcam), anti- RUNX2 (1 μg/ml; Abcam), anti-COL1A1 (1: 1000; Abcam), anti-Active (non-phosphorylated) β-catenin (1: 1000; Cell Signaling Technology), and anti-Total β-catenin (1: 1000; Cell Signaling Technology). After washing with TBST for four times, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma, United States) for 1 h at room temperature. After washing with TBST for five times, the PVDF membranes were analyzed using an enhanced chemiluminescent detection reagent (Millipore) and scanned with the BioSpectrum Imaging System (UVP, Germany).
4
Immunohistochemical Analysis of Bone Markers
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For immunohistochemistry (Spagnoli et al. 1995) (link), sections were incubated with rabbit polyclonal anti-TG2 (1:100 overnight; Covalab, Vinci-Biochem, Florence, Italy), anti-OCN (1 µg/ml for 1 h; Abcam, Cambridge, UK), anti-OPN (1 µg/ml for 1 h; Sigma-Aldrich, Milan, Italy), and anti-SOST (1 µg/ml for 1 h; Abcam, Cambridge, UK). The slides were then incubated with a goat anti-rabbit IgG peroxidase-conjugated secondary antibody (Sigma-Aldrich, Milan, Italy). After, the antigen-antibody complexes were visualized by 3-amino-9-ethylcarbazole (ScyTek, Logan, UT, USA) using Haematoxylin for counterstaining (Bei et al. 2004 (link)). Positive and negative controls were included to confirm the consistency of the analysis. Staining intensity in chondrocytes and osteocytes/osteoblasts was semiquantitatively graded using an arbitrary units system as follows: negative (0), weak and focal (0.5), weak diffuse (1), moderate (2), and high (3) (Orlandi et al. 2004 (link)). Each observation was repeated from two researchers. Inter-observer reproducibility >95 %.
5
Immunofluorescence Assay for Osteogenic Markers
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For immunofluorescence assay (Cervelli et al. 2012) (link), first passage HACs were fixed in methanol for 5 min at -20 °C and stained with rabbit polyclonal anti-TG2 (Covalab), anti-OCN (Abcam), anti-OPN (Sigma-Aldrich), and anti-SOST (Abcam; 1:20 for 1 h) and subsequently with rhodamine-labeled or fluorescein isothiocyanate-labeled antirabbit antibodies (Thermo Scientific Pierce, IL, USA), with negative controls. Nuclei were counterstained using Hoechst 33258 (Sigma-Aldrich). Cells were photographed with the Nikon ACT-1 camera controller software connected to Nikon Digital Camera DXM1200F, as reported (Cervelli et al. 2012) (link).
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