Protocol to visualize distinct motoneuron pools in adult zebrafish via injection of retrograde tracers

In adult zebrafish, slow, intermediate, and fast muscle fibers occupy distinct regions of the axial muscle, allowing the use of retrograde tracers for selective targeting of the motoneurons (MNs) innervating them. Here, we describe a protocol to label distinct MN pools and tissue processing for visualization with confocal laser microscopy. We outline the different steps for selective labeling of primary and secondary MNs together with spinal cord fixation, isolation, mounting, and imaging. For complete details on the use and execution of this protocol, please refer to Pallucchi et al. (2022)¹ and Ampatzis et al. (2013).².