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

Abstract

In this chapter, we present application of in vivo cryotechnique (IVCT) samples to luciferin-luciferase reaction (LLR) for visualization of adenosine triphosphate (ATP), an energy source for muscle contraction. The LLR was clearly detected as an intentional design of the ATP attachment to glass slides with glutaraldehyde (GA). The intensity of the light unit by LLR was correlated with the concentration of the GA-treated ATP in vitro. In tissue sections of IVCT mouse skeletal muscles followed by freeze-substitution fixation (FS) in acetone containing GA, the LLR was detected in lumino-microsope. In muscle fibers and connective tissues, the intensity of LLR was different. Differences in LLR among muscle fibers were also detected. For the IVCT-FS tissue sections, diaminobenzidine reactions were detected in type I muscle fibers and erythrocytes in capillaries. Thus, it became possible to perform microscopic evaluation of the ATP distribution in the mouse skeletal muscles with IVCT.