Combined in situ hybridization chain reaction and immunostaining to visualize gene expression in whole-mount Drosophila central nervous systems

Abstract

Methods to visualize gene expression in the Drosophila central nervous system are important in fly neurogenetic studies. In this chapter, we describe a detailed protocol that sequentially combines in situ hybridization chain reaction (HCR) and immunostaining to detect mRNA and protein expression in whole-mount Drosophila larval and adult central nervous systems. We demonstrate the application of in situ HCR in comparisons of nervous system gene expression between Drosophila species, 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.