Slides containing serial cross‐sections of thoracic descending aortic tissue or HAECs were immunostained with antiphosphorylated histone H2AX (anti‐γH2AX) antibody (Cell Signaling Technology) and then with Alexa‐488 conjugated secondary antibody. Hoechst 33342 or DAPI (4′,6‐diamidino‐2‐phenylindole) was used to stain nucleic acids and to assess the formation of senescence‐associated heterochromatin foci. Anti–LOX‐1 (Santa Cruz Biotechnology, Dallas, TX), anti‐TP53 (Cell Signaling Technology), anti‐p16INK4a (Abcam Inc., Cambridge, MA), and anti‐3‐nitrotyrosine (EMD Millipore, Darmstadt, Germany) antibodies were used to determine in situ the abundance of LOX‐1, TP53, p16INK4a, and 3‐nitrotyrosine in aortic tissue samples. Anti‐hTERT monoclonal antibody (EMD Millipore, Billerica, MA, USA) was used to detect hTERT expression in HAECs. (Klokov, MacPhail, Banath, Byrne, & Olive, 2006 ). The semi‐quantification of immunofluorescence staining is described in the Supplemental Methods.
Anti 3 nitrotyrosine
Manufactured by Merck Group
Sourced in Germany
Anti-3-nitrotyrosine is a laboratory product used for research purposes. It is an antibody that specifically binds to 3-nitrotyrosine, a post-translational modification of proteins. This product can be used in various research applications, such as immunodetection and quantification of 3-nitrotyrosine in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti tp53, by Cell Signaling Technology (1 mentions)
Anti p16ink4a, by Abcam (1 mentions)
Anti lox 1, by Santa Cruz Biotechnology (1 mentions)
Anti α actinin, by Merck Group (1 mentions)
Anti nrf2, by Santa Cruz Biotechnology (1 mentions)
Amplex red assay, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Merck Group (1 mentions)
Anti enos, by BD (1 mentions)
Anti p enos, by BD (1 mentions)
Acetylcholine, by R&D Systems (1 mentions)
Recombinant human hmgb1, by R&D Systems (1 mentions)
Phenylephrine, by Merck Group (1 mentions)
Dl homocysteine, by Merck Group (1 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Anti survivin, by Santa Cruz Biotechnology (1 mentions)
Facscan flow cytometer, by BD (1 mentions)
Anti erα, by BD (1 mentions)
Ccd camera, by Zeiss (1 mentions)
Anti phosphorylated erk1 2, by BD (1 mentions)
Microphot fluorescence microscope, by Olympus (1 mentions)
6 protocols using anti 3 nitrotyrosine
1
Immunohistochemical Analysis of Aortic Aging
2
Immunoblotting and Immunohistochemical Analysis of Cellular Signaling
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For immunoblotting analysis, the following antibodies were used: anti-AMPK and anti-3-nitrotyrosine purchased from Merck, KGaA (Darmstadt, Germany), anti-eNOS and anti-p-eNOS from BD biosciences (Heidelberg, Germany), anti-α-actinin and anti- ß-actin from Sigma-Aldrich, (Schnelldorf, Germany), and anti-Nrf-2 from Santa Cruz (Dallas, TX, USA). In addition, for immunohistochemical analysis, we used anti-endothelin-1 from ThermoFisher (Schwerte, Germany) and 3-nitrotyrosine antibody from Upstate Biotechnology (Waltham, MA, USA). Amplex Red Assay was applied to detect hydrogen peroxide (Molecular Probes, Eugene, OR, USA). Glyceryl-trinitrate (GTN; nitrolingual infusion solution, 1 mg/mL) from G. Pohl-Boskamp (Hohenlockstedt, Germany) was used for isometric tension studies. All other reagents were acquired from Sigma-Aldrich, Fluka, or Merck.
3
Quantification of HMGB-1 and eNOS in Vascular Tissue
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HMGB-1 ELISA kit (rabbit) (Cat#OKWB00408) was purchased from Aviva Systems Biology (San Diego, CA, USA); anti-eNOS/NOS Type III (Cat#610297) purified mouse monoclonal antibody and 3,3′-diaminobenzidine (DAB) substrate kit (Cat#550880) were purchased from Becton Dickinson Biosciences (VIC, Australia); unconjugated mouse monoclonal TNF-α antibody (Cat#NB600-1422) was purchased from Novus Biologicals (CO, USA); recombinant human HMGB-1 (Cat#RDS1690HMB050) was purchased from R&D Systems (Minneapolis, MN, USA); acetylcholine (Cat#A6625), DL-homocysteine (Cat#H4628), glycyrrhizic acid ammonium salt from glycyrrhiza root (licorice) (Cat#50531), phenylephrine (Cat#P1250000) and monoclonal anti-HMGB-1 (Cat#WH0003146M8) and anti-3-nitrotyrosine (Cat#N5538) antibodies produced in mouse were purchased from Sigma Aldrich (St. Louis, MO, USA); and ImmPRESS HRP goat anti-mouse IgG polymer detection kit (Cat#MP-7452-50) was purchased from Vector Laboratories (Burlingame, CA, USA).
4
Immunophenotyping of Formaldehyde-Fixed RBCs
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RBCs were fixed with 3.7% formaldehyde in PBS (pH 7.4) for 10 min at room temperature, washed in the same buffer and permeabilized with 0.5% Triton X-100 in PBS for 5 min at room temperature. After washing with PBS, samples were incubated for 30 min at 37°C with monoclonal antibodies: anti-ER-α, anti-ER-β, anti-phosphorylated ERK1/2 (BD PharMingen, San Diego, CA), anti-survivin (Santa Cruz Biotechnology) and anti-3-nitrotyrosine (Sigma Aldrich). After, all samples were washed thrice in PBS to be then incubated with secondary antibody FITC-conjugated: anti-mouse (Invitrogen, Carlsbad, CA) or anti-rabbit (Invitrogen, Carlsbad, CA). All the samples were recorded with a FACScan flow cytometer (Becton-Dickinson, Mountain View, CA, United States) equipped with a 488 United Statesnm argon laser. At least 20, 000 events were acquired. The median values of fluorescence intensity histograms were used to provide a semi-quantitative analysis. Morphometric analyses were also employed to evaluate ER-α and ER-β distribution. All samples were mounted on glass cover slips with fluorescence mounting medium (Dako) and observed by intensified video microscopy (IVM) with an Olympus Microphot fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a Zeiss CCD camera.
5
Quantifying Nitrotyrosine Levels
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The levels of nitrotyrosine were determined by Western blotting after 12% non-reducing SDS-PAGE as described before. The primary antibody was anti-3-nitrotyrosine (1:1000) from Sigma.
6
Multimodal Protein Characterization Protocol
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SDS-PAGE was performed using 4-20% gels (BioRad) and ~1 μg of pure protein input. Proteins were visualized in-gel using a Coomassie Brilliant Blue R250 staining protocol. For Western Blotting, proteins were transferred to nitrocellulose membranes (BioRad reagents) and blots were probed using the Li-COR Odyssey® system and associated reagents for infrared secondary antibodies. Primary antibodies: 8-18C5 anti-MOG (mouse monoclonal, in-house production), anti-HIS-tag (mouse monoclonal, Serotec MCA1396), anti-3-nitrotyrosine (rabbit polyclonal, Sigma, N0409) applied 1:1000.
Isoelectric focusing (IEF) was performed using Novex® systems and reagents and ~5 μg of protein input.
Isoelectric focusing (IEF) was performed using Novex® systems and reagents and ~5 μg of protein input.
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