An applicable whole-mount immunolabeling method for volume imaging of skeletal muscle

Three-dimensional mapping of skeletal muscle is particularly valuable for systematical identification and analysis of various biological components, such as nerve fibers, muscle fibers and vessels during disease and regeneration. iDISCO, as a whole-mount immunolabeling technique, provides an important tool for volume imaging of muscles, but its application has been limited to thin and small muscles of young mice owing to poor antibody penetration. In this work, we developed a modified iDISCO method and applied it to label the nerve fibers in various skeletal muscles of adult mice, including diaphragm, biceps brachii and tibialis anterior. Light-sheet microscopy was used to image biceps brachii and tibialis anterior. The results showed that the modified method could achieve uniform and complete labeling of nerve fibers within muscles, whereas original iDISCO method caused strong nonspecific signal in the surface of the muscles, and intramuscular nerve fibers were almost invisible. The modified method permitted us to trace three-dimensional nerve fibers in the muscles. This method shows potential for three-dimensional histological analysis in mouse skeletal muscle, facilitating the understanding of structural-functional relationship of skeletal muscle in physiological and pathological condition.