Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue

Keiki Ogino, Madoka Nakajima, Norio Kodama, Masayuki Kubo, Shingo Kimura, Hirohumi Nagase, Hiroyuki Nakamura

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO-2 and H2O2 or by peroxynitrite was not completely inhibited by 100mMu dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from L-tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.

Original languageEnglish
Pages (from-to)1163-1170
Number of pages8
JournalFree Radical Research
Volume36
Issue number11
DOIs
Publication statusPublished - Nov 2002
Externally publishedYes

Fingerprint

Eosinophils
Artifacts
Tissue
Dithionite
Fixatives
Antibodies
Ovalbumin
Tyrosine
Anti-Idiotypic Antibodies
3-nitrotyrosine
Nitration
Peroxynitrous Acid
periodate-lysine-paraformaldehyde
Therapeutics
Gastric Mucosa
Peroxidase
Epitopes
Assays
Proteins
Western Blotting

Keywords

  • Artifact
  • Eosinophils
  • Immunohistochemistry
  • Nitrotyrosine
  • PLP fixative

ASJC Scopus subject areas

  • Biochemistry

Cite this

Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue. / Ogino, Keiki; Nakajima, Madoka; Kodama, Norio; Kubo, Masayuki; Kimura, Shingo; Nagase, Hirohumi; Nakamura, Hiroyuki.

In: Free Radical Research, Vol. 36, No. 11, 11.2002, p. 1163-1170.

Research output: Contribution to journalArticle

Ogino, K, Nakajima, M, Kodama, N, Kubo, M, Kimura, S, Nagase, H & Nakamura, H 2002, 'Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue', Free Radical Research, vol. 36, no. 11, pp. 1163-1170. https://doi.org/10.1080/1071576021000016427
Ogino, Keiki ; Nakajima, Madoka ; Kodama, Norio ; Kubo, Masayuki ; Kimura, Shingo ; Nagase, Hirohumi ; Nakamura, Hiroyuki. / Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue. In: Free Radical Research. 2002 ; Vol. 36, No. 11. pp. 1163-1170.
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