High resolution three-dimensional imaging: Evidence for cell cycle reentry in regenerating skeletal muscle

Newts and other urodele amphibians can replace lost structures including limbs, providing a vertebrate model for the study of regeneration of complex tissues. The composite of different cell and tissue types in the limb, however, presents a challenge for their imaging in three-dimensions (3D) at cellular level resolution. To observe myofibers in vivo without distortion, we developed a streamlined protocol whereby 80 μm thick cryosections are mounted on slides, processed for immunohistochemistry, imaged using confocal microscopy and z-stacks rendered in 3D. This methodology enabled precise in situ rendering of regenerating muscle, demonstrating cell cycle reentry of nuclei within the myofiber syncytium. The high resolution imaging of muscle or comparable tissue types as intact 3D entities in the context of extracellular and intracellular molecules allows for the determination of signaling and cell response pathways, making this method useful for studies that attempt to characterize rare physiological events in vivo.