Visualizing and quantifying oxidized protein thiols in tissue sections: A comparison of dystrophic mdx and normal skeletal mouse muscles

Tomohito Iwasaki, Jessica R. Terrill, Tea Shavlakadze, Miranda D. Grounds, Peter G Arthur

Research output: Contribution to journalArticleResearchpeer-review

13 Citations (Scopus)

Abstract

Reactive oxygen species (ROS) are not only a cause of oxidative stress in a range of disease conditions but are also important regulators of physiological pathways in vivo. One mechanism whereby ROS can regulate cell function is by modification of proteins through the reversible oxidation of their thiol groups. An experimental challenge has been the relative lack of techniques to probe the biological significance of protein thiol oxidation in complex multicellular tissues and organs. We have developed a sensitive and quantitative fluorescence labeling technique to detect and localize protein thiol oxidation in histological tissue sections. In our technique, reduced and oxidized protein thiols are visualized and quantified on two consecutive tissue sections and the extent of protein thiol oxidation is expressed as a percentage of total protein thiols (reduced plus oxidized). We tested the application of this new technique using muscles of dystrophic (mdx) and wild-type C57Bl/10Scsn (C57) mice. In mdx myofibers, protein thiols were consistently more oxidized (19 ± 3%) compared with healthy myofibers (10 ± 1%) in C57 mice. A striking observation was the localization of intensive protein thiol oxidation (70 ± 9%) within myofibers associated with necrotic damage. Oxidative stress is an area of active investigation in many fields of research, and this technique provides a useful tool for locating and further understanding protein thiol oxidation in normal, damaged, and diseased tissues.

Original languageEnglish
Pages (from-to)1408-1416
Number of pages9
JournalFree Radical Biology and Medicine
Volume65
DOIs
Publication statusPublished - 2013
Externally publishedYes

Keywords

  • Free radicals
  • Histochemistry
  • Murine
  • Muscular dystrophy
  • Oxidative stress
  • Protein thiol
  • Reactive oxygen species
  • Skeletal muscle
  • Sulfhydryl

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