Immunohistochemical Artifact for Nitrotyrosine in Eosinophils or Eosinophil Containing Tissue
| Autor: | Hiroyuki Nakamura, Masayuki Kubo, Norio Kodama, Hirohumi Nagase, Shingo Kimura, Keiki Ogino, Madoka Nakajima |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Ovalbumin
chemical and pharmacologic phenomena Dithionite Biochemistry Immunoenzyme Techniques chemistry.chemical_compound Western blot immune system diseases medicine Humans Tyrosine Nitrogen Compounds Chromatography High Pressure Liquid Peroxidase biology medicine.diagnostic_test Nitrotyrosine Periodic Acid Hydrogen Peroxide General Medicine respiratory system Eosinophil Oxidants nervous system diseases Eosinophils medicine.anatomical_structure chemistry Gastric Mucosa biology.protein Immunohistochemistry lipids (amino acids peptides and proteins) Artifacts Oxidation-Reduction Peroxynitrite |
| Zdroj: | Free Radical Research. 36:1163-1170 |
| ISSN: | 1029-2470 1071-5762 |
| DOI: | 10.1080/1071576021000016427 |
| Popis: | 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 NO2- and H2O2 or by peroxynitrite was not completely inhibited by 100 mM 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|