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Visualization of ATP with Luciferin-Luciferase Reaction in Mouse Skeletal Muscles Using an “In Vivo Cryotechnique”

Published online by Cambridge University Press:  12 October 2012

N. Terada ,
Y. Saitoh ,
S. Saitoh ,
N. Ohno ,
K. Fujishita ,
S. Koizumi  and
S. Ohno
N. Terada*
Affiliation:
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan Department of Occupational Therapy, School of Health Sciences, Shinshu University School of Medicine, Matsumoto-city, Nagano 390-8621, Japan
Y. Saitoh
Affiliation:
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan
S. Saitoh
Affiliation:
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan
N. Ohno
Affiliation:
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan
K. Fujishita
Affiliation:
Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan
S. Koizumi
Affiliation:
Department of Neuropharmacology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan
S. Ohno
Affiliation:
Department of Anatomy and Molecular Histology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo-city, Yamanashi 409-3898, Japan
*
*Corresponding author: E-mail: nobuot@shinshu-u.ac.jp
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Abstract

Adenosine triphosphate (ATP) is a well-known energy source for muscle contraction. In this study, to visualize localization of ATP, a luciferin-luciferase reaction (LLR) was performed in mouse skeletal muscle with an “in vivo cryotechnique” (IVCT). First, to confirm if ATP molecules could be trapped and detected after glutaraldehyde (GA) treatment, ATP was directly attached to glass slides with GA, and LLR was performed. The LLR was clearly detected as an intentional design of the ATP attachment. The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. Next, LLR was evaluated in mouse skeletal muscles with IVCT followed by freeze-substitution fixation (FS) in acetone-containing GA. In such tissue sections the histological structure was well maintained, and the intensity of LLR in areas between muscle fibers and connective tissues was different. Moreover, differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine (DAB) reactions were clearly detected in type I muscle fibers and erythrocytes in capillaries, which demonstrated flow shape. Thus, it became possible to perform microscopic evaluation of the numbers of ATP molecules in the mouse skeletal muscles with IVCT, which mostly reflect living states.

Keywords

in vivo cryotechniqueskeletal muscleATPluciferine-luciferase reactionfreeze-substitution fixationdiaminobenzidine reaction
Type
Techniques and Equipment Development
Information
Microscopy and Microanalysis , Volume 18 , Issue 5 , October 2012 , pp. 1030 - 1036
Copyright
Copyright © Microscopy Society of America 2012

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