Protocol for multiplexed RNAscope-based imaging of mRNAs in whole-mount adult Drosophila brains

Visualizing the expression of mRNAs using traditional in situ hybridization is often hampered by obstacles including weak signal, high background, and poor probe specificity. Here, we present a protocol utilizing RNAscope (ACD) to overcome these obstacles and detect multiple types of mRNAs simultaneously in whole-mount adult Drosophila brains. We further describe how mRNAs can be reliably quantified in any cells that can be targeted by common binary expression systems such as Gal4/UAS and labeled by immunohistochemistry. For complete details on the use and execution of this protocol, please refer to De et al.1