Briefly, paraffin sections were deparaffinized in xylene, rehydrated in graded alcohols and antigens were retrieved in citrate buffer. Endogen peroxidase was blocked using a peroxidase-blocking reagent (Dako) and non-specific binding sites were blocked by the Protein Block Serum Free solution (Dako). The slides were then incubated with the primary antibody “anti-human RANKL (70525, R&D systems) or anti-OPG (polyclonal, Abcam)”. Immunoperoxidase staining was performed using the EnVision system (Dako) according to the supplier's recommendations. Colorimetric detection was completed with diaminobenzidine (Dako) for 5 min. Slides were then counterstained with hematoxylin. RANKL and OPG expression was evaluated, as previously described, by a semi-quantitative score of the intensity and extent of the staining according to an arbitrary scale.59 (link)
Anti opg
Manufactured by Abcam
Sourced in United States, China
Anti-OPG is a lab equipment product designed to detect and measure the levels of Osteoprotegerin (OPG) in biological samples. OPG is a protein that plays a crucial role in regulating bone remodeling and mineral metabolism. The product is intended for research use only and provides a reliable and accurate method for quantifying OPG concentrations in various experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Anti rankl, by Abcam (3 mentions)
Anti ocn, by Abcam (2 mentions)
Anti runx2, by Abcam (2 mentions)
Protein block serum free solution, by Agilent Technologies (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Gapdh, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protein estimation kit, by Merck Group (1 mentions)
Anti bmp2, by Abcam (1 mentions)
Anti p jak2, by Abcam (1 mentions)
Anti p stat3, by Abcam (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Anti tlr2, by Abcam (1 mentions)
Anti il 1β, by Abcam (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti tlr4, by Abcam (1 mentions)
Anti rank, by Abcam (1 mentions)
Anti rankl, by Novus Biologicals (1 mentions)
5 protocols using anti opg
1
Immunohistochemical Evaluation of RANKL and OPG
2
Femur Protein Expression Analysis
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The right femur tissues were homogenized in RIPA buffer using a homogenizer and the homogenate was centrifuged at 12,000 g for 15 min at 4°C. Total proteins of the supernatant were measured using the Bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific). Protein samples were separated by 15% SDS-polyacrylamide gels, and then transferred onto a PVDF membrane (Roche, USA). After blocking with 5% skim milk for 1 h at room temperature, the membrane was hybridized with the following primary antibodies overnight at 4°C: anti-OPG (1:1,000; Abcam, USA), anti-RANKL (1:1,000; Abcam), anti-RunX2 (1:1,000; Abcam), anti-OCN (1:1,000; Abcam), and GAPDH (1:1,000; Abcam). The membrane was washed with TBST and subsequently incubated with horseradish peroxidase-labeled secondary antibody for 45 min at room temperature. Visualization was performed with chemiluminescence detection reagent (ECL, USA). The relative protein levels were analyzed with ImageJ image analysis software (National Institutes of Health, USA) and normalized to GAPDH.
3
Quantifying Bone Protein Expression
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Total proteins were isolated using RIPA buffer from both cells and bone tissues then analyzed with a protein estimation kit (Sigma Aldrich, USA). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels were used to separate 10 µg of proteins and transferred to PVDF membranes. Prior to incubating the membranes with antibodies they were blocked with non-fat milk (5%). The antibodies used were anti-Runx2 (1:500 Abcam), anti-RANKL (1:500 Abcam), anti-BMP2 (1:500 Abcam), anti-OPG (1:500, Abcam), anti-p-STAT3 (1:500 Abcam) and anti-p-JAK2 (1:500 Abcam). The antibodies were incubated with membranes for 12 hours at 4 °C. The blots were then incubated with secondary antibodies for 1 hour at room temperature and visualized with the aid of chemiluminescent reagent and densitometric analysis was done by image reading software (Bio-Rad, USA). The relative expression of bands was done using actin as a loading control.
4
Western Blot Analysis of Osteogenic Markers
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Cells were harvested, lysed with lysis buffer (ASPEN, Wuhan, China), and
centrifuged at 16,200 g for 10 min. Total proteins in the supernatant were
measured using a BCA protein assay kit (ASPEN, Wuhan, China). Total proteins
were extracted from MC3T3-E1 cells. The protein at 30 µg was resolved by 10%
SDS-PAGE gels (ASPEN, Wuhan, China). After electrophoresis, the proteins were
transferred onto nitrocellulose membrane (Millipore, USA). The membranes were
blocked with 5% nonfat milk (ASPEN, Wuhan, China) at room temperature for 1 h.
The samples were probed with anti-GAPDH (1:10,000; Abcam, UK), anti-IL-1β
(1:1000; Abcam, UK), anti-TNF-α (1:500; Abcam, UK), anti-RANK (1:1000; Abcam,
UK), anti-RANKL (1:500; Novusbio, Shanghai, China), anti-OPG (1:1000; Abcam,
UK), anti-OCN (1:500; Abcam, UK), anti-TLR2 (1:1000; Abcam, UK), and anti-TLR4
(1:500; Abcam, UK) Abs. HRP-conjugated goat anti-rabbit IgG (1:10000; ASPEN,
Wuhan, China) was used for detection.
centrifuged at 16,200 g for 10 min. Total proteins in the supernatant were
measured using a BCA protein assay kit (ASPEN, Wuhan, China). Total proteins
were extracted from MC3T3-E1 cells. The protein at 30 µg was resolved by 10%
SDS-PAGE gels (ASPEN, Wuhan, China). After electrophoresis, the proteins were
transferred onto nitrocellulose membrane (Millipore, USA). The membranes were
blocked with 5% nonfat milk (ASPEN, Wuhan, China) at room temperature for 1 h.
The samples were probed with anti-GAPDH (1:10,000; Abcam, UK), anti-IL-1β
(1:1000; Abcam, UK), anti-TNF-α (1:500; Abcam, UK), anti-RANK (1:1000; Abcam,
UK), anti-RANKL (1:500; Novusbio, Shanghai, China), anti-OPG (1:1000; Abcam,
UK), anti-OCN (1:500; Abcam, UK), anti-TLR2 (1:1000; Abcam, UK), and anti-TLR4
(1:500; Abcam, UK) Abs. HRP-conjugated goat anti-rabbit IgG (1:10000; ASPEN,
Wuhan, China) was used for detection.
5
Immunohistochemical Analysis of OPG and RANKL
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Paraffin-embedded heads from 2 samples from both groups were sectioned in the coronal plane at 4 μm thickness and mounted onto slides. Sections were dipped in xylene to remove the paraffin and dehydrated through a graded alcohol series. In order to prevent endogenous peroxidase activity, sections were incubated with 3% hydrogen peroxide in methanol for 30 min at room temperature following sodium citrate antigen unmasking treatment for 20 min at 96 °C. Five percent normal goat serum in PBS-Triton (0.3%) was used for blocking at room temperature. The following antibodies were used: anti-OPG and anti-RANKL (1:200, rabbit polyclonal antibody, Abcam) in 5% normal Bovine Serum at 5%, overnight at 4 °C. After that, the slides were treated with peroxidase staining (Sigma-Aldrich) and developed with diaminobezidine tetrahydrochloride (DAB-Vector Laboratories, Burlingame, CA, USA). The slides were then counterstained with Harris hematoxylin or methyl green and mounted with Entellan (Merck Millipore, São SP, Brazil) in order to visualize nuclei of the cells. Negative controls were obtained by omitting the primary antibody of each reaction. The slides were photographed under a Nikon Eclipse TE300 microscope (Nikon Company, Melville, NY, USA).
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