Conclusions
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.
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. Conclusions: 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.