The red flour beetle, Tribolium castaneum, is an emerging model system well suited to the study of embryonic brain development and evolution (see Chapters 11 and 13 ). Brain genesis is driven by specific gene products whose expression underlies a tight spatiotemporal control. Therefore, the analysis of gene expression in time and space provides valuable insights into the molecular mechanisms that govern brain development. Since Tribolium-specific antibodies are scarce, fluorescent RNA in situ hybridization is the method of choice to determine the dynamics of individual gene expression. We have modified common RNA in situ protocols to facilitate the concomitant detection of two gene-specific expression patterns (double fluorescent RNA in situ). In addition, we describe a procedure which combines fluorescent single RNA in situ and immunostaining with gene-specific antibodies. Conventional in situ using RNA probes that are complementary to mature mRNAs often produce diffuse signals. We demonstrate that RNA in situ probes complementary to intronic gene sequences facilitate single cell resolution because the fluorescent signal is restricted to the nucleus. We believe our protocols can be adapted easily to suit the analysis of brain development in other insect species.