Quantifying Kidney Nitrosylated Oxidation

We evaluated the levels of 3-NTY as a marker of kidney nitrosylated oxidation products caused by formation of peroxynitrite. Five micrometer sections of the kidney were initially quenched of endogenous peroxidase and incubated with 1:200 rabbit polyclonal anti-3-NTY antibody overnight (Chemicon, Temecula, CA) [40 (link), 41 (link)]. Sections were washed and incubated with appropriate secondary antibody and signals visualized by diaminobenzidine (DAB) chromogen system (DAKO, Carpinteria, CA). Using a 50i Nikon microscope, five randomly selected 10× bright-field images from each section were captured with a CoolSNAP cf camera. Signal intensities of brownish color, which is indicative of the 3-NTY level, were quantified by MetaVue software.

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Habibi J., Aroor A.R., Das N.A., Manrique-Acevedo C.M., Johnson M.S., Hayden M.R., Nistala R., Wiedmeyer C., Chandrasekar B, & DeMarco V.G. (2019). The combination of a neprilysin inhibitor (sacubitril) and angiotensin-II receptor blocker (valsartan) attenuates glomerular and tubular injury in the Zucker Obese rat. Cardiovascular Diabetology, 18, 40.

Publication 2019

diaminobenzidine (DAB) chromogen system CoolSNAP cf camera

Anti antibody Antibody Chromogen Kidney Microscope Peroxidase Peroxynitrite Rabbit

Corresponding Organization :

Other organizations : University of Missouri, The University of Texas Health Science Center at San Antonio, University of Missouri Health System, University of Missouri Hospital, Harry S. Truman Memorial Veterans' Hospital

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Variable analysis

independent variables

Use of 1:200 rabbit polyclonal anti-3-NTY antibody

dependent variables

Levels of 3-NTY as a marker of kidney nitrosylated oxidation products caused by formation of peroxynitrite

control variables

Five micrometer sections of the kidney
Quenching of endogenous peroxidase
Incubation time of overnight for the anti-3-NTY antibody
Use of appropriate secondary antibody
Visualization of signals by diaminobenzidine (DAB) chromogen system
Capturing of five randomly selected 10× bright-field images from each section using a CoolSNAP cf camera
Quantification of signal intensities of brownish color (indicative of 3-NTY level) by MetaVue software

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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