Immunostaining is used to visualize the spatiotemporal expression pattern of developmental control genes that regulate the genesis and specification of the embryonic and larval brain of Drosophila. Immunostaining uses specific antibodies to mark expressed proteins and allows their localization to be traced throughout development. This method reveals insights into gene regulation, cell-type specification, neuron and glial differentiation, and posttranslational protein modifications underlying the patterning and specification of the maturing brain. Depending on the targeted protein, it is possible to visualize a multitude of regions of the Drosophila brain, such as small groups of neurons or glia, defined subcomponents of the brain's axon scaffold, or pre- and postsynaptic structures of neurons. Thus, antibody probes that recognize defined tissues, cells, or subcellular structures like axons or synaptic terminals can be used as markers to identify and analyze phenotypes in mutant embryos and larvae. Several antibodies, combined with different labels, can be used concurrently to examine protein co-localization. This protocol spans over 3-4 days.