Combinedin situhybridization chain reaction and immunostaining to visualize gene expression in whole-mountDrosophilacentral nervous systems

Methods to visualize gene expression in theDrosophilacentral nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combinesin situhybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mountDrosophilalarval and adult central nervous systems. We demonstrate the application ofin situHCR in comparisons of nervous system gene expression betweenDrosophilaspecies, and in the validation of single-cell RNA-Seq results in the fly nervous system. Our protocol provides a simple, robust, multiplexable, and relatively affordable means to quantitatively visualize gene expression in the nervous system of flies, facilitating its general use in fly neurogenetic studies.