Protocol for whole-brain immunolabeling, clearing, and light-sheet imaging of C-FOS in the pigeon

Advancements in tissue clearing and light sheet microscopy have provided new ways to study neural circuits dedicated to sensory processing in a diverse range of animal species. Here, we present a modified iDISCO+ protocol for whole-brain immunolabeling with the neural activity marker C-FOS in the pigeon brain. We describe steps for whole-brain pre-processing, bleaching, and immunostaining. We then detail procedures for obtaining ultramicroscopic light-sheet images in cleared pigeon brains. For complete details on the use and execution of this protocol, please refer to Nordmann et al.¹.