Three-dimensional visualization of intramuscular innervation in intact adult skeletal muscle by a modified iDISCO method

Three-dimensional visualization of the innervation in skeletal muscles is helpful for understanding the morphological structure and function. iDISCO, a whole-mount immunolabeling and clearing technique, provides a valuable tool for volume imaging of intramuscular nerve fibers but suffers from the nonspecific staining caused by the anti-mouse secondary antibody when using the murine primary antibody. We developed a modified iDISCO method by introducing pretreatment of ScaleCUBIC-1 reagent, termed m-iDISCO. The m-iDISCO method could eliminate the nonspecific staining and achieve uniform and complete labeling of nerve fibers in various muscles with mouse anti-neurofilament primary antibody. Combining the m-iDISCO method with light-sheet microscopy enabled us to visualize the innervation of adult mouse tibialis anterior and trace the nerve fibers from extramuscular branches to intramuscular terminal branches. This method represents an effective alternative for studying the innervation of intact skeletal muscles in health and disease.