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

Yusha Li,Tingting Yu,Dan Zhu,Jianyi Xu,Jingtan Zhu

doi:10.1117/1.nph.7.1.015003

Abstract

.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.

Highlights

Three-dimensional (3-D) visualization of intramuscular innervation can be valuable for understanding morphological structures and functions of the skeletal muscles.[1]
Various tissue optical clearing methods, such as 3DISCO, CUBIC, SWITCH, and CLARITY, have been combined with whole-mount immunostaining for labeling and imaging of large-volume tissues.[12,13,14,15,16] iDISCO, a simple method with no need of customized setup, enables facile detection of immunolabeled structures throughout intact embryos and dense adult tissues.[17]
The followed anti-mouse secondary antibody can abundantly bind to the dense sarcolemma and the fascia, especially for the ones with increased thickness in adult mouse skeletal muscles [Fig. 2(a)], which will substantially hinder penetration of antibodies and reduce the effective antibody binding

Summary

Introduction

Three-dimensional (3-D) visualization of intramuscular innervation can be valuable for understanding morphological structures and functions of the skeletal muscles.[1] Immunostaining on sectioned tissue is a powerful approach for labeling molecules of interest and revealing cellular distribution in many biological research studies, but it often requires extensive labor and time for 3-D reconstruction with potential artifacts. To circumvent these issues, whole-mount immunostaining methods have been developed to label intact large tissues, but it is still quite challenging to achieve uniform and complete labeling due to the difficulty of antibody penetration.[2,3,4,5,6]. Various tissue optical clearing methods, such as 3DISCO, CUBIC, SWITCH, and CLARITY, have been combined with whole-mount immunostaining for labeling and imaging of large-volume tissues.[12,13,14,15,16] iDISCO, a simple method with no need of customized setup, enables facile detection of immunolabeled structures throughout intact embryos and dense adult tissues.[17]

Objectives

Methods

Results

Conclusion