Total protein was extracted from bone tissues using the bone tissue protein extraction kit (BestBio, China) following the manufacturer's instructions. Concentrations of proteins were measured using a BCA protein assay kit (Thermo Fisher Scientific, IL, USA). For western blot analysis, proteins were separated using SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, MA, USA). Subsequently, PVDF membranes carrying target peptides were blocked in 5% skim milk (Beyotime, China) and then incubated with primary antibodies against RANKL (1 : 1,000; PA5-110268, Invitrogen, CA, USA), OPG (1 : 1,000; ab73400, Abcam, UK), FoxO3a (1 : 1,000; ab23683, Abcam), Wnt1 (1 : 3,000; PA5-85217, Invitrogen), β-catenin (1 : 5,000; ab73400, Abcam), and GAPDH (1 : 10,000; ab181602, Abcam) at 4°C overnight. Next, membranes were incubated with secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (1 : 2,000; ab6721, Abcam) for 2 h. Band intensity of protein peptide was detected using the enhanced chemiluminescence system with the ImagePro Plus software.
Ab73400
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab73400 is a laboratory instrument used for protein detection and quantification. It is designed to measure the concentration of specific proteins in biological samples. The core function of this product is to provide accurate and reliable results for protein analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ab9957, by Abcam (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ab8245, by Abcam (3 mentions)
Ab19027, by Abcam (3 mentions)
Ripa buffer, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Ab6721, by Abcam (2 mentions)
Zli 9018, by Zhongshan Jinqiao Biotechnology (2 mentions)
Ab45039, by Abcam (2 mentions)
Ab110304, by Abcam (2 mentions)
Ab93876, by Abcam (2 mentions)
Sc52950, by Santa Cruz Biotechnology (2 mentions)
Optical microscope, by Olympus (2 mentions)
Horseradish peroxidase labeled goat anti rabbit igg and anti mouse igg, by Abcam (2 mentions)
3 3 diaminobenzidine (dab), by Solarbio (2 mentions)
Ab239607, by Abcam (2 mentions)
Pentobarbital sodium, by Solarbio (1 mentions)
Ab14933, by Abcam (1 mentions)
Ab93926, by Abcam (1 mentions)
Ab23981, by Abcam (1 mentions)
23 protocols using ab73400
1
Bone Tissue Protein Extraction and Western Blot Analysis
2
Immunohistochemical Analysis of Osteoprotegerin and APT1
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The 2 consecutive paraffined sections of bone tissues were collected for routine dewaxing and hydration, incubated with 3% H2O2 at room temperature for 5 min to block endogenous peroxidase activity, and incubated with 10% goat serum at room temperature for 10 min. After washing with phosphate buffer saline (PBS), the sections were incubated with primary antibodies anti-Osteoprotegerin (1:2000, ab73400, Abcam, Cambridge, MA, USA) or anti-APT1 (1:500, K20033, Biolab, Beijing, China) at 4 °C overnight, followed by incubation with the second antibody IgG (1:200, ab6721, Abcam) for 30 min at room temperature and with streptomycin peroxidase for 20 min at room temperature. Subsequently, the samples were added with freshly prepared DAB developing solution, restrained with hematoxylin, turned blue with tap water, dehydrated, and sealed. An optical microscope was applied for observation and photography. The percentage (%) of APT1 positive antigen-expressing cells in each visual field was analyzed using ImageJ software, and 5 non-overlapping visual fields were randomly selected from each slice, and the results were averaged.
3
SaOS-2 Cell Culture and Reagent Characterization
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SaOS-2 cell was kept in the laboratory. GKC (20180521) was obtained from Guizhou Wei Kang Zi Fan Pharmaceutical Co. Ltd. (Guiyang, China). Jiegu-Qili tablet was obtained from Hunan Jin Sha Pharmaceutical Co. Ltd. (Changsha, China). Pentobarbital Sodium (6900183) was purchased from Beijing Solarbio Science & Technology Co. Ltd. Penicillin G Sodium (170907) was from Lukang Pharmaceutical Co. Ltd. (Jining, China). ALP (A059-2), Pi (C006-3), and Ca (C004-2) were supplied by Nanjing Jiancheng Biotechnology Co. Ltd. (Nanjing, China). Primers of ALP, COL-I, OTC, Osterix, RUNX2, BMP2, OPN, OPG, RANKL, and GAPDH were obtained from Shanghai Generay Biotech Co. Ltd. (Shanghai, China). Antibodies against RUNX-2 (ab23981), OPG (ab73400), BMP2 (ab14933), RANKL (ab9957), β-catenin (ab6302), Smad4 (ab40759), GAPDH (ab8245), and GSK3β (ab93926) were from Abcam (Cambridge, England). DKK1 (PHC9214), Noggin (PHC1506), antibodies against Smad1/5 (PA5-80036), and p-Smad1/5 (MA5-15124) were obtained from Thermo fisher (American). p-GSK3β (Ser9, 9336S) antibody was got from Cell Signaling Technology (American). All other reagents used in the present study were of analytical grade.
4
Immunohistochemical Analysis of OPG and RANKL
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Paraffin-embedded sagittal sections of decalcified mandible tissue were sliced 5 μm thick, deparaffinized in xylene two times and dehydrated with gradient concentrations of ethanol. To block endogenous peroxidase activity, the slides were incubated with 3% hydrogen peroxide for 15 min and blocked with 5% BSA for 30 min at room temperature to prevent nonspecific binding. Then, the slides were incubated with the primary antibodies anti-OPG (1:50, ab73400; Abcam) and anti-RANKL (1:200, ab9957; Abcam) at 4 °C overnight. After being immersed in a secondary antibody for 1 h at room temperature, the sections were washed with PBS (pH 7.2–7.6) three times for 5 min each. Then, 3,3’- diaminobenzidine (DAB) (ZLI-9018; Zhongshan Jinqiao Biotechnology Co.) was used for the colour reaction, which was stopped with distilled water, followed by counterstaining with haematoxylin. Finally, dehydration and sealing were performed with neutral gum. Slides were photographed using a light microscope (Olympus, Tokyo, Japan), and the fractional (%) stained area was calculated using Image-Pro Plus software.
5
Western Blot Analysis of Osteoblast Markers
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The total proteins of MC3T3-E1 cells or BMSCs were extracted and lysed with RIPA buffer (Beyotime, China), and protein concentration was detected using a BCA protein assay kit (Solarbio, China). Then, a 20 μg total protein sample was loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for separation and transferred to a PVDF membrane. After being blocked with 5% skimmed milk, the membrane was incubated at 37°C with the primary antibodies against caspase-3 (ab13847, Abcam), caspase-9 (ab32539, Abcam), SIRT1 (ab110304, Abcam), BMP-2 (ab214821, Abcam), RANKL (ab45039, Abcam), OPG (ab73400, Abcam), IκBα (ab32518, Abcam), p-IκBα (AF2002, Affinity), p65 (ab207297, Abcam), p-p65 (AF2006, Affinity), IKKα (ab32041, Abcam), p-IKKα (AF3013, Affinity), NFATc1 (ab2796, Abcam), and cathepsin K (ab19027, Abcam) at 4°C overnight. After that, the membrane was further incubated with the secondary antibody at room temperature for 1 h. Then, the enhanced chemiluminescence (ECL) reagent (Solarbio, China) was used to visualize the protein bands, and the relative band density was semiquantified with the ImageJ software.
6
Western Blot Analysis of Bone Metabolism Markers
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Protein was extracted by using RIPA buffer (Beyotime, Shanghai, China). 10% SDS-PAGE was used to separate the protein samples. Then, the protein was transferred into PVDF membrane. After blocking with 5% nonfat milk, the protein was incubated with OPG (ab73400, 1 : 1000, Abcam), RANK (ab200369, 1 : 1000, Abcam), RANKL (ab9957, 1 : 1000, Abcam), STAT3 (ab119352, 1 : 1000, Abcam), and GAPDH (ab8245, 1 : 2000, Abcam) primary antibodies at 4°C overnight. Then, peroxidase-conjugated secondary antibody (ab7090, 1 : 2000, Abcam) was added to incubate the protein for 1 h at room temperature. The protein bands were developed by using the ECL system (Pierce, Rockford, IL, USA). The density of the bands was analyzed utilizing the Quantity One software.
7
Quantifying Pancreatic Insulin Expression
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Isolated mouse pancreata were fixed using Mildform (Wako) overnight at 4°C and embedded in paraffin (Sakura Finetek Japan). Sections of 4 μm were treated with 0.2% Triton-X100/PBS for 5 min and incubated with boiled 0.01 M citrate buffer, pH 6.0, for 20 min. Prior to application of primary antibody, sections were treated with Blocking Solution A from the Histofine MOUSESTAIN kit (Nichirei). Primary antibodies, including anti-OPG rabbit polyclonal antibody at 5.0 μg/ml (ab73400, abcam), anti-insulin mouse monoclonal antibody IN-05 at 1.0 μg/ml (ab7760, abcam), and anti-glucagon mouse monoclonal antibody K79bB10 at 4.4 μg/ml (ab10988, abcam) were applied overnight at 4°C. After washing with PBS, cells were stained with Alexa 488-conjugated goat anti-mouse IgG or Alexa 594-conjugated goat anti-rabbit IgG (Molecular Probes) for 1 hr at room temperature. After washing, sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories) and observed under an LSM710 confocal fluorescence microscope (Carl Zeiss) to determine signal localization or under a DMi6000B microscope (Leica) to measure signal intensity. The average signal intensity was measured by freehand region of interest (ROI) using Tissue Studio (Definiens) software to quantify insulin expression levels in pancreatic β-cells.
8
Immunohistochemical Analysis of Macrophage and Bone Markers
9
Osteogenic Protein Expression Analysis
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AS fibroblasts were lysed with RIPA buffer (Beyotime Shanghai, China) to extract total protein. After quantified using BCA Kit (Beyotime), equal amounts of proteins were separated with SDS-PAGE gel and transferred onto PVDF membranes (Millipore). Then, the membranes were blocked with 5% non-fat milk and incubated with antibodies against runt-related transcription factor 2 (RUNX2; 1:1,000, ab76956, Abcam, Cambridge, MA, United States), osteocalcin (OCN; 1:500, ab93876, Abcam), osteoprotegerin (OPG; 1:1,000, ab73400, Abcam), SOST (1:1,000, ab85799, Abcam) or β-actin (1:5,000, ab8227, Abcam) at 4°C overnight. After incubated with secondary antibodies against Goat Anti-Rabbit IgG (1:10,000, ab205718, Abcam) or Goat Anti-Mouse IgG (1:5,000, ab205719, Abcam), protein signals were visualized using enhanced chemiluminescence (ECL) reagent (Yeasen). The experiment was performed three biological replicates.
10
Immunohistochemical Analysis of Bone Biomarkers
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Immunostaining for tumor necrosis factor-α (TNF-α), RANKL and osteoprotegerin (OPG) was performed on 4-μm decalcified right distal femoral sections. Briefly, all sections were deparaffinized in xylene, rehydrated, and washed in PBS. The sections were then heated for 20 min in citrate buffer (pH 6.0) at 95°C and incubated with an anti-tumor necrosis factor-α (TNF-α) monoclonal antibody (1:100, catalog number: ab199013, Abcam, United States), anti-RANKL polyclonal antibody (1:200, catalog number: BA1323, Wuhan Boster, China), and anti-Osteoprotegerin (OPG) polyclonal antibody (1:500, catalog number: ab73400, Abcam, United States) overnight at 4°C. Phosphate-buffered saline (PBS) was used as a negative control. Then, the sections were incubated with secondary antibodies for 30 min at 26°C. 3,3′-Diaminobenzidine (DAB) staining and hematoxylin counterstaining were performed to visualize the expression of biomarkers. Immunohistochemical analysis was performed with Image-Pro Plus 6.0 software to evaluate immunostaining.
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