Frozen cross sections of aortic valve embedded in OCT were stained for immunofluorescence as previously described (22 (link)). Primary antibodies used were as follows: osterix (1:500, Abcam), phospho-SMAD1/5/8 (1:100, Cell Signaling), phospho-SMAD3 (1:100, Abcam), and 3-nitrotyrosine (1:100, Abcam). For all staining assays, negative controls were used to confirm validity and consistency of the staining pattern. All sections were stained with Alexa Fluor 647™ (produced in donkey and specific to the respective species secondary; all used at 1:500 dilution, Invitrogen) and mounted in Prolong Gold Anti-fade with DAPI (Invitrogen). Subsequently, a Zeiss LSM 780 confocal microscope set at 20 × magnification was used to focus and capture images of the base portions of the aortic valve leaflet. Following image acquisition, sections were analyzed using IMAGEJ software (NIH).
3 nitrotyrosine
Manufactured by Abcam
Sourced in United States
3-nitrotyrosine is a modified amino acid that can be used as a biomarker for oxidative stress and nitrosative damage. It is a stable end-product of the reaction between nitric oxide, superoxide, and tyrosine residues in proteins.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Phospho and total gsk3β, by Cell Signaling Technology (2 mentions)
Recombinant human egf, by Merck Group (2 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharide lps, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Osterix, by Abcam (1 mentions)
Phospho smad1 5 8, by Cell Signaling Technology (1 mentions)
Phospho smad3, by Abcam (1 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
P enos, by Abcam (1 mentions)
Total enos, by Abcam (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
13 protocols using 3 nitrotyrosine
1
Immunofluorescence Analysis of Aortic Valve Calcification
2
Protein Isolation and Western Blot Analysis
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Protein was isolated from liver tissue after incubation in RIPA Lysis Buffer (Beyotime Institute of Biotechnology) supplemented with protease inhibitor cocktail (Sigma-Aldrich, Dublin, Ireland) for 1 h on ice. After centrifugation (14000 × g, 4 °C, 15 min), the supernatants were collected for protein concentration measurement using a Bicinchoninic Acid Protein Assay Kit (Thermo Fisher Scientific). Denatured protein was separated by SDS-PAGE (Invitrogen, Carlsbad, CA, United States) and transferred to nitrocellulose membranes. After blocking with 50 mL/L non-fat milk in Tris-buffered saline with Tween (TBST; 10 mmol/L Tris-HCl, 0.5 mL/L Tween 20 and 0.15 mol/L NaCl, pH 7.2) for 3 h, the membranes were incubated with primary antibodies against β-actin (1:1000; Abcam, Cambridge, MA, United States), total eNOS (1:1000; Abcam), p-eNOS (1:500; Abcam), 3-nitrotyrosine (1:1000; Abcam) for 15 h at 4 °C. Blots were washed in TBST and incubated with appropriate horseradish-peroxidase-linked anti-mouse or anti-rabbit secondary antibodies (goat anti-mouse IgA for total eNOS, goat anti-rabbit IgG for p-eNOS and goat anti-mouse IgG2a for 3-nitrotyrosine; all supplied by Abcam) for 1 h at room temperature. Enhanced chemiluminescence was conducted using an ECL kit (Pierce Biotechnology, Rockford, IL, United States).
3
Protein Extraction and Western Blot Analysis
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Liver tissues were homogenized in a lysis buffer (100 mM NaCl, 50 mM Tris at pH 7.4, 1 mM EDTA, 1% Triton X-100 with protease inhibitor and phosphatase inhibitor cocktails, Roche) and denatured in a 2% SDS sample buffer (50 mM Tris at pH 6.8, 2% SDS, 100 mM dithiothreitol and 10% glycerol) for 10 min at 100 °C. The extracted proteins were separated by SDS–polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene fluoride membrane. The membranes were blocked with 5% (w/v) nonfat dried milk, probed with an antibody and detected with a Visualizer Kit (WBKLS0500, Millipore, Burlington, MA, USA). The following antibodies were used for Western blotting: S-sulfonated cysteine (ADI-OSA-820; Enzo Life Sciences, Ann Arbor, MI, USA), 3-nitrotyrosine (ab61392, Abcam, Cambridge, UK) and Gapdh (MAB374, Millipore, Burlington, MA, USA).
4
Mitochondrial Dysfunction and Oxidative Stress
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Gn-Rb1 was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Antibodies against gp91phox, SOD2, 3-nitrotyrosine, 4 hydroxynonenal, DRP1, DRP1 (phospho S637), DRP1 (phospho S616), Mitofusin 2, OPA1, Fis1, Histone H3, caspases-3, cleaved caspases-3, and VDAC1 were obtained from Abcam (Cambridge, MA, USA). GAPDH, Bax, and Bcl-2 were obtained from Cell Signaling Technology (Beverly, MA, USA). Nrf2, keap1, NQO1, HO1, Ndufs1, Ndufv1, Ndufs6, Ndufs4, Ndufv2, and Ndufa12 were obtained from Abmart (Shanghai, China). Dihydroethidium (DHE) wasobtained from Beyotime (Jiangsu, China).
5
Protein Separation and Western Blot Analysis
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Proteins were separated and transferred to an Immobilon membrane (Millipore, Bedford, MA) as previously described47 (link). After electrotransfer, the filters were incubated with the appropriate monoclonal antibody against β-actin (Sigma-Aldrich, Alcobendas, Spain) or polyclonal antibody against inducible nitric oxide synthase (iNOS ), C/EBP homologous protein (CHOP ), Agouti related protein (AGRP ), ghrelin , neuropeptide Y (NPY ), melanin concentrating hormone (PMCH ), CRP , albumin , IL-6 , p tyr 1138 Ob/Rb , p tyr 985 Ob/Rb , p tyr Stat3 , p ser Stat3 , SOCS3 , PTP1B (Santa Cruz Biotechnology, Santa Cruz, CA), pserIRS -1 and ptyrIRS -1 (Cell Signaling Technology, Danvers, MA), leptin (Thermo Fisher Scientific, Waltham, MA), TNF-α , and 3-nitrotyrosine (Abcam plc, Cambridge, UK). Signals were detected using the ECL Western Blotting Detection Reagent (Amersham Ibérica, Madrid, Spain).
6
Comprehensive Antibody Protocol for NEC
7
Protein Expression Analysis by Western Blotting
8
Comprehensive Antibody Protocol for NEC
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Antibodies were obtained from the following sources: p65 subunit of NF-κB, phospho and total GSK3β (Cell Signaling); PCNA (Santa Cruz); 3-nitrotyrosine (Abcam), actin (Genscript), bromodeoxyuridine (BrdU, Novus Biosciences); DAPI (Invitrogen). TUNEL (Roche Applied Science) was performed according to the manufacturer’s instructions as in 22 (link). Appropriate secondary antibodies for immunohistochemistry and SDS-PAGE were obtained from Molecular Probes and Jackson ImmunoResearch Laboratories. Cetuximab (Dose: 100 μg/day i.p. for 3 days prior to experiments) was a generous gift of Jennifer Grandis (University of Pittsburgh, Pittsburgh, PA). Human Recombinant EGF was from EMD Millipore (400 ng/mL media in vitro, 0.5ng per μL of NEC formula in vivo). Lithium chloride (LiCl) from J.T. Baker (100μM). Lipopolysaccharide (LPS) (Escherichia coli 0111:B4 purified by gel-filtration chromatography, >99% pure) was obtained from Sigma-Aldrich and concentrations used were those that we have demonstrated to be present in mice and humans with NEC (50 μg/mL for cells, 5mg/kg for all mice3 with the exception of the TLR4ΔIEC-OVER mice who are sensitive to LPS and required a decreased dosage (2.5 mg/kg).
9
Endothelial Cell Growth and Treatment
10
Comprehensive Protein Analysis Protocol
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Western blot analysis was measured as described previously [29 (link),30 (link)]. Tissue and cell extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with the appropriate primary antibodies against PKM2 (Cell signaling technology, 4053), pAKT (Cell signaling technology, 13038), total AKT (Cell signaling technology, 9272), pSTAT3 (Cell signaling technology, 9145), total STAT3 (Cell signaling technology, 9139), NOX4 (Novus, NB110-58849), SOD2 (Cell signaling technology, 13141), NOX2 (Abcam, ab129068), catalase (Cell signaling technology, 14097), 3-nitrotyrosine (Abcam, ab61392), CDK5 (Cell signaling technology, 12134), GSKβ (Cell signaling technology, 12456), FBW7 (BETHYL, A301-721A), Myc (Cell signaling technology, 2276), Flag (Cell signaling technology, 14793), HA (Cell signaling technology, 3724), and β-Actin (Santa Cruz, sc-47778) at 4 °C overnight. The membrane was extensively washed and then incubated with appropriate secondary antibody. Proteins were visualized by using an available chemiluminescence kit (GE Healthcare).
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