Anti-Nitrotyrosine Antibodies for Immunohistochemistry

Abstract

Nitrotyrosine is an important marker for the formation of peroxynitrite and possibly other reactive nitrogen species derived from nitric oxide in vivo (1). Pathological conditions can substantially increase the production of nitric oxide, yet this molecule itself does not generally yield nitration of tyrosine residues in proteins when added to biological samples (1,2). However nitric oxide reacts at near diffusion-limited rates with superoxide (O(2) (-)) to form the strong oxidant peroxynitrite (ONOO(-)) (3). Nitration on the 3-position of tyrosine is a major product of peroxynitrite attack on proteins (4,5). Certainly, small amounts of nitrotyrosine can be produced in vivo by other mechanisms (6), but peroxynitrite is by far the most efficient mechanism for nitrating tyrosine under biologically relevant conditions with natural antioxidants and alternative targets present.

Evidence for Reactive Nitrogen Species Formation in the Gingivomucosal Tissue

“…Following pre-treatment with 20% sucrose/PBS, 40-pm-thick sections were cut on a freezing microtome, rinsed in PBS, and treated with 0.3% H202 to quench endogenous peroxidase activity. The freefloating sections were reacted against a primary rabbit anti-3nitrotyrosine polyclonal antiserum (1:1000 in PBS with 1% normal goat serum for 48 hrs at 4°C;Viera et al, 1999Viera et al, , 2000. Following rinses in PBS, the sections were incubated with a biotinylated goat anti-rabbit IgG (1:200), then with the avidinbiotin-peroxidase complex (1:100, Vectastain ABC Kit) for 2 hrs at room temperature.…”

“…Immunohistochemical controls involved omitting either the primary or the secondary antibody from the incubation media or mixing the working dilution of the primary antibody with 10 mM 3-nitrotyrosine (for 3 hrs in PBS adjusted to pH 7.4 ) prior to the immunohistochemical staining (Viera et al, 1999(Viera et al, , 2000.…”

A Free Radical Scavenger Ameliorates Teratogenic Activity of a DNA Hypomethylating Hematological Therapeutic

“…Nitrones act on a variety of pathways to change the redox state through scavenging ROS or reactive nitrogen species (ROS/RNS) but they are also involved in controlling signaling transduction and gene induction [12,18] through NO-releasing properties [19]. Nitric oxide reacts with superoxide (O 2 – ) to form the strong oxidant peroxynitrite (ONOO–), and nitration on the three positions of tyrosine represents a major product of peroxynitrite attack on proteins that may disrupt phosphorylation that is crucial for signal transduction pathways [20].…”

Temporal patterns of tyrosine nitration in embryo heart development

“…Tissue sections from the hearts of embryos from different ages (E12-E19) and of newborns were immunostained as previously described [22]. Briefly, paraffin sections of the specimens were immunostained with a polyclonal anti-3-nitrotyrosine antibody [22,23], eNOS and iNOS antibodies (BD Biosciences, Franklin Lakes, NJ).…”

“…Briefly, paraffin sections of the specimens were immunostained with a polyclonal anti-3-nitrotyrosine antibody [22,23], eNOS and iNOS antibodies (BD Biosciences, Franklin Lakes, NJ). Tissue sections were preincubated with 0.3% hydrogen peroxide in absolute methanol, washed in PBS, blocked with 10% goat serum, and incubated with the primary antibody.…”