Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

The remodeling of the mitochondrial network occurs in physiological stress and pathological conditions. However, the effects of mitochondrial remodeling in a skeletal muscle remain incompletely understood. This protocol enables the quantification of changes of mitochondria in live skeletal muscle enhancing our comprehension of the interplay between mitochondrial alterations and cellular function.

Working with live skeletal muscles present challenges due to fiber contraction during isolation and sensitivity to image during confocal scanning. This protocol describes an isolation process using a relaxation solution, enabling the analysis of viable muscle fibers for mitochondrial assessment via confocal microscopy. Our protocol addresses the lack of workflows to analyze quantitatively mitochondrial networks in skeletal muscle fibers.

The steps described here allow users to take more advantage of images obtained by confocal microscopy using image processing by thresholding and fast Fourier analysis to achieve quantification of mitochondrial density and distribution. We are interested in the study of skeletal muscle alteration, particularly mitochondrial dynamics and mitochondrial structural remodeling during adversity and how can they be modulated through intervention such as exercise and intermediate fasting.