A total of 100 mg peritoneal tissue was collected from each rat and lysed with 500 μl of cold protein lysate RIPA (Solarbio) buffer containing protease inhibitor (0.4 mM PMSF, 1 mM Iodo, 1 μM Pepstatin A). Centrifugation was performed at 14000 rpm at 4°C for 10 min, after which the supernatant was collected. Protein concentration was determined by the BCA method. Samples were loaded as 50 μg of protein in 5X loading buffer. The protein sample was separated by SDS-PAGE, transferred to a PVDF membrane (Millipore), and blocked with 5% skimmed milk solution at room temperature for 1 h. Membranes were then incubated with primary antibodies [Gremlin1 (1:1000), ab157576; Abcam; BMP7 (1:1000), ab56023; Abcam); TGFβ1 (1:1000), ab92486; Abcam; and β-Actin (1:1000), PAB36265; Bioswamp] at 4 °C overnight. On the following day, the membranes were washed and incubated with Goat anti-Rabbit secondary antibodies [(1:20000), SAB43714; Bioswamp]. After washing with TBST, the membrane was visualized using a gel imaging analyzer (FluorChem FC2 Imaging System; Alpha Innotech). Image analysis was performed using the AlphaView software (Alpha Innotech).
Ab56023
Manufactured by Abcam
Sourced in United States, United Kingdom
Ab56023 is a laboratory equipment product manufactured by Abcam. It serves as a core function in scientific research and analysis. The detailed description of its intended use and capabilities is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Ab39973, by Abcam (2 mentions)
Dfc 3200 camera, by Leica camera (2 mentions)
Ab150077, by Abcam (2 mentions)
Bicinchoninic acid assay, by Thermo Fisher Scientific (2 mentions)
Ab92486, by Abcam (1 mentions)
Fluorchem fc2 imaging system, by Bio-Techne (1 mentions)
Alphaview software, by Bio-Techne (1 mentions)
Pab36265, by Bioswamp (1 mentions)
Sab43714, by Bioswamp (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bicinchoninic acid assay, by CWBIO (1 mentions)
Ab34718, by Abcam (1 mentions)
Ab78422, by Abcam (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Ab6276, by Abcam (1 mentions)
Ab32391, by Abcam (1 mentions)
Ab16051, by Abcam (1 mentions)
Ab13420, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab75745, by Abcam (1 mentions)
9 protocols using ab56023
1
Western Blot Analysis of Fibrosis Markers
2
Investigating Epigenetic Regulators in Tissue Samples
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A radioimmunoprecipitation assay buffer with a protease inhibitor cocktail (Solarbio, Beijing, China) and 1 mmol/L phenylmethylsulphonyl fluoride was used for the extraction of the total cellular protein from the snap-frozen tissues. A bicinchoninic acid assay (CWBIO, Beijing, China) was applied to the determination of the total protein concentration. Afterward, 10% sodium dodecyl sulfate sulphate polyacrylamide gel electrophoresis was used to separate samples with 100 μg of protein, followed by electrophoretic transfer to nitrocellulose membranes. A blocking buffer (5% skim milk, 0.1% Tween 20®, and 0.01 M tris-buffered saline) was then used to block the membranes at room temperature for 2 hours. Anti-KDM5C (1:100 dilution, ab34718, Abcam, USA), anti-BMP-7 (1:150 dilution, ab56023, Abcam, USA), and anti-BMPRII (1:150 dilution, ab78422, Abcam, USA) were used to incubate the membranes overnight at 4°C. β-actin (1:120,000 dilution, ab6276, Abcam, USA) was used to express all results. Secondary fluorescent antibodies (1:5000 dilution, Odyssey CIX, Lincoln, USA) were then used to incubate the membranes for 2 hours at room temperature, after which an infrared laser scanning imaging system (Odyssey CIX, Lincoln, USA) was used to measure the fluorescence intensity. Based on the ratio of the internal target protein, analysis of protein expression for all target genes was conducted.
3
Quantifying Osteogenic Markers in MC3T3-E1 Cells
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After the induction of high glucose and treatment of catalpa, proteins in MC3T3-E1 cells were extracted with RIPA lysis buffer (Beyotime, China) and protein concentration was determined by Bradford method. Each hole was loaded with 30μg protein, separated with the SDS-PAGE gel for electrophoresis and transferred to nitrocellulose membranes. The 5% skim milk solution was prepared with the solvent of PBST, which was used to seal the nitrocellulose membranes at room temperature for 2h. Then, primary antibodies including RUNX2 (ab192256;Abcam), Collagen I (ab34710;Abcam), OCN (ab13420;Abcam), BMP4 (ab39973;Abcam), BMP7 (ab56023;Abcam), KDM7A (A14692;ABclonal), β-catenin (ab16051;Abcam), pGSK3β (ab75745;Abcam), GSK3β (ab32391;Abcam), histone (ab1220;Abcam) and GAPDH (ab9485;Abcam) (dilution,1:1000) were incubated with nitrocellulose membranes at 4°C for 12h. PBST was used to wash the nitrocellulose membranes 3 times with 10 min/time. HRP-labeled secondary antibody was incubated with the nitrocellulose membranes at room temperature for 1h. After the nitrocellulose membranes were fully washed, they were then treated with ECL chemiluminescent solution from Millipore. The Bio-Rad gel imaging analysis system was used to collect and analyze the bands.
4
Western Blot Analysis of Ad-MSCs and BMP-7-MSCs
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Ad-MSCs and BMP-7-MSCs were used for Western blot analysis. Briefly, the cells were washed twice with DPBS, and sonicated in lysis buffer (150 mM sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 50 mM Tris at pH 7.5, 2 mM ethylenediaminetetraacetic acid) on ice for 30 min. Lysates were cleared by centrifugation (10 min at 13,000× g and 4 °C), and protein concentrations were determined using the Bradford method [37 (link)]. Equal amounts of protein (15 μg) were resolved by electrophoresis on 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. Membrane blots were washed with TBST (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20), blocked with 5% skim milk for 1 h, and incubated with the appropriate primary antibodies at the recommended dilutions. The antibodies used included antibodies against actin (A3853, Sigma-Aldrich), BMP-7 (ab56023, Abcam, Cambridge, UK). The primary antibodies (1:1000) were diluted in TBST. The membrane was then washed, and primary antibodies were detected with goat anti-rabbit IgG or goat anti-mouse IgG conjugated to horseradish peroxidase (1:5000, Invitrogen, Carlsbad, CA, USA). Bands were visualized using enhanced chemiluminescence (Invitrogen).
5
Quantifying Bone Morphogenetic Proteins
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Total protein from tissues or cells was extracted using lysis buffer provided in the phosphorylated Smad1 ELISA kit. Protein concentration was determined using the bicinchoninic acid assay (ThermoFisher Scientific, Waltham, MA, USA), and equal loads of reduced protein samples were electrophoretically separated on NuPAGE 4–12% Bis-Tris Gel (Invitrogen) and then transferred to a polyvinylidene difluoride (PVDF) membrane using the iBlot Dry Blotting System (Invitrogen). Primary antibodies directed against BMP-2 (ab14933; Abcam), BMP-4 (ab39973; Abcam), BMP-7 (ab56023; Abcam), BMP-9 (ab35088; Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST5174; Cell Signaling Technology, Danvers, MA, USA), total Smad1 (CST6944; Cell Signaling Technology) and phosphorylated Smad1/5 (CST9516; Cell Signaling Technology) were incubated at 4 °C overnight, followed by horseradish peroxidase–conjugated secondary antibody (GE Healthcare Life Sciences, Marlborough, MA, USA) incubation. Western blots were developed using SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific) and visualized using a FOTO/Analyst 1 Fx CCD imaging system (Fotodyne, Hartland, WI, USA).
6
Histological Analysis of Muscle Tissue
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Muscle tissues were fixed in methanol and embedded in paraffin. For histology, sections (9 μm thick) were collected on Colorfrost Plus Microscope Slides (ThermoFisher Scientific) and rehydrated, and hematoxylin and eosin (H&E) staining was performed according to a routine Harris Hematoxylin and Eosin protocol. Immunohistochemistry (IHC) was performed according to protocols described for the Vectastain Elite ABC Kit (Vector Laboratories Inc., Burlingame, CA, USA). Images were taken using a CKX41 microscope (Olympus, Tokyo, Japan) equipped with a DFC 3200 camera (Leica, Wetzlar, Germany). For immunofluorescence staining, tissue sections were blocked in 10% bovine serum albumin for 20 min and then incubated overnight at 4 °C with mixed primary antibodies against BMP-7 (ab56023; Abcam) and CD68 (ab53444; Abcam). On the second day, sections were incubated with mixed secondary antibodies against rabbit IgG (ab150077; Abcam) and rat IgG (ab150158; Abcam) at room temperature for 1 h. After washing, the slides were mounted with 4′,6-diamidino-2-phenylindole (DAPI)-containing mounting medium (Vector Laboratories). Slides were viewed using an inverted IX81 microscope (Olympus) equipped with a Retiga EXi cooled CCD camera (Qimaging, Surrey, bc , Canada) and MetaMorph software (Molecular Devices, San Jose, CA, USA).
7
BMP-7 and Gremlin1 Pathway Analysis
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Trizol reagent, the reverse transcription (RT) kit, and semiquantitative real-time PCR kit were purchased from Takara (Takara, Japan). Rabbit polyclonal antibodies specific for BMP-7 (ab56023, Abcam, USA) and gremlin1 (ab140010, Abcam, USA), Smad1/5/8 (sc-7965, Santa Cruz, Canada), phosphorylated (p)-SMAD1/5/8 (SMAD1, 5-phospho-Ser463/465, and SMAD8 phospho-Ser 426/428 antibodies; 13820, Cell Signaling Technology, Canada), and p-Smad2/3 (YP0362, Immunoway Biotechnology, China) were used in this study. Secondary antibodies were obtained from Jackson (Jackson, USA). The TGF-β1 and FN enzyme-linked immunosorbent assay (ELISA) kits were purchased from Boster (EK0515, Boster, Wuhan, China). Low-glucose Dulbecco’s modified Eagle medium (DMEM) was purchased from Hyclone (Hyclone, USA). Penicillin/streptomycin and trypsin were obtained from North China Pharmaceutical (NCPC, China). BCAAs were purchased from Sigma-Aldrich Company (USA). Glucose was purchased from China Otsuka Pharmaceutical Company (Otsuka, China). Mannitol was purchased from Shandong JinYang Pharmaceutical (Shandong JinYang Pharmaceutical, China).
8
Histochemical Analysis of Calcification
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Hematoxylin and Eosin (H&E) staining was performed according to routine Harris H&E protocol. For alizarin red staining, slides were stained with alizarin red solution (Alfa Aesar, Haverhill, MA, USA) for 45 s. The extent of calcification was evaluated by the ratio (%) of the calcium staining area to the total area, which could be calculated with the Image J version 1.5.0 software (National Institutes of Health). There were totally four region of interest every group and blinding was not performed. For Immunofluorescence (IF) staining, sections were blocked in 10% bovine serum albumin for 30 min and then incubated overnight at 4°C with mixed primary antibodies against ALP (ab83250, Abcam), OSX (ab22552, Abcam) and BMP-7 (ab56023, Abcam). Sections were incubated with fluorescein isothiocyanate-conjugated secondary antibody (ab150077, Abcam) for 1 h, then mounted with 4′, 6-diamidino-2-phenylindole (DAPI) on second day. The images were taken by an Olympus CKX41 microscope with a Leica DFC 3200 camera.
9
Immunohistochemical Detection of BMP-2 and BMP-7
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BMP-2 and BMP-7 detection was performed on paraffin sections rehydrated, permeabilised with 0.1 % Triton X-100 in PBS and incubated in 0.3 % (v/v) H 2 O 2 for 10 min. Antigen retrieval steps were performed in 10 mM sodium citrate buffer (pH 6.0) for 30 min at 95 °C. Then, the sections were blocked in 5 % (w/v) bovine serum albumin (BSA) in PBS for 30 min and subsequently incubated for 2 h at room temperature with 2.5 μg/mL rabbit-anti-human BMP-2 (C43125; LifeSpan BioSciences, Nottingham, UK) or 2 μg/mL rabbit-anti-human BMP-7 (ab56023; Abcam) in PBS/5 % BSA. Negative control labellings were performed with 2.5 and 2 μg/mL non-immunised rabbit IgG in PBS (X0903; Dako). Subsequently, samples were incubated with 1 μg/mL goat antirabbit horseradish peroxidase (P0448; Dako) in PBS/5 % BSA for 60 min at room temperature. The staining was developed with 3,3'-diaminobenzidine (DAB). Counterstaining was performed with Mayer's haematoxylin.
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