Estimating relative carbonyl levels in muscle microstructures by fluorescence imaging |
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Authors: | Juan Feng Marian Navratil LaDora V Thompson Edgar A Arriaga |
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Institution: | (1) Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455, USA;(2) Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA;(3) Department of Physical Medicine and Rehabilitation, University of Minnesota, Minneapolis, MN 55455, USA |
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Abstract: | The increase in the levels of protein carbonyls, biomarkers of oxidative stress, appears to play an important role in aging
skeletal muscle. However, the exact distributions of carbonyls among various skeletal muscle microstructures still remain
largely unknown, partly owing to the lack of adequate techniques to carry out these measurements. This report describes an
immunohistochemical approach to determine the relative abundance of carbonyls in the intermyofibrillar mitochondria (IFM),
the subsarcolemmal mitochondria (SSM), the cytoplasm, and the extracellular space of skeletal muscle. These morphological
features were defined by labeling the nucleus, the Z-lines, and mitochondria. Carbonyls were detected by derivatization with
dinitrophenylhydrazine followed by labeling with an Alexa 488-labeled anti-dinitrophenyl primary antibody. Alexa 488 fluorescence
(green) in different fiber microstructures was used to estimate the relative abundance of carbonyls. On the basis of the samples
examined, preliminary results suggest that the most dramatic age-related changes in carbonyl levels occur in the extracellular
space, followed in a decreasing order by SSM, IFM, and the cytoplasm. These observations were confirmed in the soleus and
semimembranosus muscles composed predominantly of type I and type II fibers, respectively. This approach could easily be extended
to the investigation of carbonyl levels in other muscles (composed of mixed skeletal muscle fiber types) or other tissues
in which protein carbonyls are present.
Figure Imaging of Labeled Carbonyls in Rat Skeletal Muscle |
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Keywords: | Protein carbonyls Aging Skeletal muscle microstructures Fluorescence microscopy Mitochondria |
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