A Protocol for Dissection, Immunostaining and Mounting of the Drosophila Visual System

The Drosophila visual system is a powerful model for exploring how neuronal diversity is generated and integrated into functional neural circuits - a central question in developmental neuroscience. It comprises the retina and four distinct neuropils: the lamina, medulla, lobula, and lobula plate, which together form a highly organized structure that processes visual input from the retina and relays it to the higher centers of the brain. During development, the visual system undergoes significant remodeling. During larval development, neuroblasts are progressively generated from the neuroepithelium and divide asymmetrically to produce diverse neuronal subtypes by integrating spatial and temporal patterning programs. These neurons then differentiate and form precise synaptic connections that build the functional circuits of the adult visual system. These dynamic processes can be reliably studied using immunostaining across larval, pupal, and adult stages. Herein, we provide a detailed, reproducible protocol for dissecting and immunostaining the developing and adult Drosophila visual system. We primarily focus on representative imaging of larval neuroblasts to highlight spatiotemporal patterning and neuronal specification, and to visualize adult neuropil morphology. This provides a robust framework for visualizing the developing Drosophila visual system.