Past Issue

Volume 20, Number 2, Summer 2018, Serial Number: 78, Pages: 132-137

Skeletal Muscle CLARITY: A Preliminary Study of Imaging The Three-Dimensional Architecture of Blood Vessels and Neurons


Wenli Zhang, B.Sc, 1, #, Shaohua Liu, B.Sc, 2, #, Weichen Zhang, B.Sc, 3, #, Wei Hu, M.Sc, 4, 5, #, Min Jiang, Ph.D, 4, Amin Tamadon, D.V.M., Ph.D., 4, 5, *, Yi Feng, M.D., Ph.D., 4, 5, *,
Department of Digestive Diseases of Huashan Hospital, Fudan University, Shanghai, China
Department of Sports Medicine, Huashan Hospital, Fudan University, Shanghai, China
Department of Nephrology, Huashan Hospital, Fudan University, Shanghai, China
State Key Laboratory of Medical Neurobiology and Institute of Brain Science, Brain Science Collaborative Innovation Center, Fudan University, Shanghai, China
Department of Integrative Medicine and Neurobiology, School of Basic Medical Sciences, Shanghai Medical College, Institutes of Integrative Medicine of Fudan University, Shanghai, China
*Corresponding Address: State Key Laboratory of Medical Neurobiology and Institute of Brain Science Brain Science Collaborative Innovation Center Fudan University Shanghai 200032 China Emails:amintamaddon@yahoo.com,fengyi17@fudan.edu.cn

These authors contributed equally to this work.

Abstract

Objective

Passive CLARITY is a whole-tissue clearing protocol, based on sodium dodecyl sulfate (SDS) clearing, for imaging intact tissue containing transgenic or immunolabeled fluorescent proteins. In this study, we present an improved passive CLARITY protocol with efficient immunolabeling without the need for electrophoresis or complex instrumentation.

Materials and Methods

In this experimental study, after perfusion of C57BL/6N mice with phosphate-buffered saline (PBS) and then with acrylamide-paraformaldehyde (PFA), the quadriceps femoris muscle was removed. The muscle samples were post-fixed and degassed to initiate polymerization. After removing the excess hydrogel around the muscle, lipids were washed out with the passive CLARITY technique. The transparent whole intact muscles were labeled for vessel and neuron markers, and then imaged by confocal microscopy. Three-dimensional images were reconstructed to present the muscle tissue architecture.

Results

We established a simple clearing protocol using wild type mouse muscle and labeling of vasculatures and neurons. Imaging the fluorescent signal was achieved by protein fixation, adjusting the pH of the SDS solution and using an optimum temperature (37˚C) for tissue clearing, all of which contributed to the superiority of our protocol.

Conclusion

We conclude that this passive CLARITY protocol can be successfully applied to three-dimensional cellular and whole muscle imaging in mice, and will facilitate structural analyses and connectomics of large assemblies of muscle cells, vessels and neurons in the context of three-dimensional systems.