Isolated cells were cultured for 2 days on chamber slides washed with PBS and fixed with 3% paraformaldehyde in PBS for 15 minutes. The cells were permeabilized with 0.1 % Triton-X in PBS for 10 minutes, and incubated with 0.3 % H2O2 for 10 minutes at room temperature. Cells were blocked with secondary-host serum for 20 minutes and incubated with rabbit polyclonal antibodies specific for osteocalcin (1:250; Abbiotec, CA, USA), sclerostin (1:150; Abcam, MA, USA) and podoplanin (1:100; Abbiotic, CA, USA) for 90 minutes at room temperature. After washing the cells three times, cells were then incubated with secondary biotinylated horse universal antibody, ABC reagent and DAP (ABC kit, Vector Lab, CA, USA), respectively, according to manufacturer instructions. Cells were counterstained with hematoxylin for 1 minute, dehydrated and cover slipped. liver tissue was used as positive control for osteocalin and sclerostin white lunch tissue was used as positive controls for podoplanin (Fig 2 ). Negative controls were used by staining our isolated cells (Fig 3 A ) and MLO-Y4 (Fig 3 B ) without primary antibody to exclude non-specific signals and false positive results.
Sclerostin
Manufactured by Abcam
Sourced in United States
Sclerostin is a protein that functions as a negative regulator of bone formation. It plays a key role in the Wnt signaling pathway, which is involved in the regulation of bone metabolism. Sclerostin inhibits the activity of osteoblasts, the cells responsible for bone formation.
Automatically generated - may contain errors
Lab products found in correlation
Rankl, by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Abc reagent, by Vector Laboratories (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by ZSGB-BIO (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
β actin, by Abcam (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Enhanced chemiluminescence reagent, by Keygen Biotech (1 mentions)
Ecl advance blocking agent, by GE Healthcare (1 mentions)
Sod 110, by Stressgen (1 mentions)
P erk erk, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Las 3000 mini, by Fujifilm (1 mentions)
Mmp13, by Abcam (1 mentions)
C fos, by Santa Cruz Biotechnology (1 mentions)
Col x, by Abcam (1 mentions)
β catenin, by Merck Group (1 mentions)
Polink 2 plus polymer hrp detection kit, by ZSGB-BIO (1 mentions)
7 protocols using sclerostin
1
Immunohistochemical Analysis of Bone Cells
2
Analysis of Osteogenic Regulators in MC3T3-E1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MC3T3-E1 cells were seeded at 3 × 105/well in six-well cell culture plate. After 48 h of treatment with Ti CM and SR, cells were washed twice with cold PBS and then lysed with RIPA buffer containing protease inhibitor and phosphatase inhibitor. The lysates were centrifuged, and the supernatant was collected to determine the protein concentration using a BCA Protein Assay Kit (KeyGEN BioTECH). Total cell protein was separated by 12% SDS-PAGE and then transferred to a PVDF membrane. After 2 h of blocking with the Tris-tween buffer containing dry milk, samples were incubated with the primary antibody overnight at 4°C. Primary antibodies used in the present study were β-actin (1:1000), RANKL (1:1000), OPG (1:500), sclerostin (SOST, 1:1000) (Abcam), β-catenin (1:1000) (Cell Signaling Technology, MA, U.S.A.). Finally, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (ZSGB-bio, Beijing, China) for 2 h at room temperature. The protein on the membrane was detected as a fluorescent band by the enhanced chemiluminescence reagent (KeyGEN BioTECH).
3
Bone Marrow Protein Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tibia was thoroughly flushed bone marrow cells with saline. The flushed tibia was homogenized using a handy sonicator and then centrifuged at 12,000 × g for 30 minutes. The supernatant was subsequently assayed for the protein concentration using the DC Protein Assay Kit (BioRad, Hercules, CA). Equal amounts (20 μg) of total protein were subjected to 10% SDS-PAGE and electroblotted onto a PVDF membrane. The membranes were blocked in 2% blocking reagent (ECL Advance Blocking Agent, GE Healthcare) and probed with antibodies against Cx43 (1:1,000; Cell signaling), RANKL (1:1,000; abcam), SOD2 (1:2,000; SOD-110; StressGen), Sclerostin (1:500; abcam), p-ERK, ERK (1:1,000; Cell signaling), GAPDH (1:1,000; Cell signaling) and ACTIN (1:2,000; Cell signaling). The signals were detected using the ECL system (ECL plus, GE Healthcare) and a luminoimage analyzer LAS-3000 mini (Fuji Film, Tokyo, Japan).
4
Immunohistochemistry Analysis of Bone Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paraffin sections of 4-week-old tibiae were rehydrated and digested in 0.1% trypsin for 10 min at the room temperature, and then treated with 3% H2O2 for 20 min. Sections were incubated with primary antibodies in PBS overnight at 4 °C. Col-X, MMP13, MMP9, cathepsin K, OPG, DKK1, and sclerostin antibodies were obtained from Abcam (Cambridge, MA, USA). Axin1 and β-catenin antibodies were obtained from Sigma (St. Louis, MO, USA). NFATc-1 and c-Fos antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Negative control sections were incubated with IgG (Beyotime Biotechnology, Shanghai, China). A Polink-2 plus polymer HRP detection kit (PV-9001, ZSGB-BIO, Shanghai, China) was used for incubation with secondary antibody and horseradish peroxidase (HRP)-streptavidin.
5
Western Blot Analysis of Sclerostin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After incubation with or without strontium at 12.5 μM for 7 days, 40 μg of total protein was separated by SDS-PAGE, using a gradient gel ((10–12%), Bio-Rad Laboratories), transferred to nitrocellulose membrane, and analyzed by immunoblotting using the chemiluminescence (Santa Cruz, CA, USA). The primary antibodies used were sclerostin (Abcam, MA, USA, 1 : 500) or GAPDH (Santa Cruz, CA, USA, 1 : 1000), peroxidase-conjugated anti-mouse IgG (Santa Cruz, CA, USA, 1 : 1000). ImageJ software was applied to compare the intensity of protein bands to control through quantifying.
6
Antioxidant and Neurological Marker Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Assay kits for superoxide dismutase (SOD) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), mouse malonaldehyde (MDA) and mouse glutathione peroxidase (GSH-px) ELISA kits were purchased from Beijing Chengzhikewei Biotechnology Co., Ltd. (Beijing, China), and radioimmunoprecipitation assay (RIPA) lysis buffer and the BCA protein detection kit were purchased from Epizyme Biotech (Shanghai, China). Antibodies against nuclear factor E2-related factor 2 (Nrf2, 1:1000), heme oxygenase-1 (HO-1, 1:1000), p21 (1:1000), p53 (1:1000), Tau (1:1000), BDNF (1:1000), GAPDH (1:1500), β-actin (1:1500), and anti-rabbit horseradish peroxidase (HRP)-conjugated IgG were purchased from Cell Signal Technique (Boston, MA, USA). Antibodies against RUNX2 (1:1000), sclerostin (1:1000), and β-catenin (1:1000) were purchased from Abcam (Cambridge, UK).
7
Immunofluorescence Staining of Cellular Structures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were fixed with paraformaldehyde and then washed with PBST (PBS-Triton X100) containing normal goat serum (Thermo Fisher Scientific) for 30 min to avoid non-specific binding of antibodies. We incubated the fixed cells with primary antibodies against Sclerostin (Abcam) or Src (Cell Signaling) at 4°C for half a day. After rinsing with PBST, we applied secondary antibodies (Alexa Fluor 546 Immunoglobulin G, Thermo Fisher Scientific) and DAPI (Thermo Fisher Scientific) at room temperature for 2 h. For visualization of F-actin, the cells were incubated with Alexa Fluor 488 phalloidin (Thermo Fisher Scientific). Finally, the stained cells were washed in PBST, followed by mounting using Antifade Reagent (Prolong Gold, Thermo Fisher Scientific). Fluorescent images were obtained under a confocal microscope (FV1000D-IX81, Olympus) and processed using the Adobe Photoshop 10.0 software. For quantitative analysis of dendritic cell processes, dendrites with length of more than 20 μm were counted[22 ]. The angle of cell processes was analyzed as a reference of substrate collagen orientation.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!