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 • Optimized whole-brain clearing protocol for avian brain tissue • Techniques for utilizing the neuronal activity marker C-FOS after sensory stimulation • Steps for light-sheet imaging and visualization of C-FOS in the whole pigeon brain Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.