Live Imaging in Planarians: Immobilization and Real-Time Visualization of Reactive Oxygen Species.

Imaging of living animals allows the study of metabolic processes in relation to cellular structures or larger functional entities. To enable in vivo imaging during long-term time-lapses in planarians, we combined and optimized existing protocols, resulting in an easily reproducible and inexpensive procedure. Immobilization with low-melting-point agarose eliminates the use of anesthetics, avoids interfering with the animal during imaging-functionally or physically-and allows recovering the organisms after the imaging procedure. As an example, we used the immobilization workflow to image the highly dynamic and fast-changing reactive oxygen species (ROS) in living animals. These reactive signaling molecules can only be studied in vivo and mapping their location and dynamics during different physiological conditions is crucial to understand their role in developmental processes and regeneration. In the current protocol, we describe both the immobilization and ROS detection procedure. We used the intensity of the signals together with pharmacological inhibitors to validate the signal specificity and to distinguish it from the autofluorescent nature of the planarian.