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

## TL;DR

This paper introduces a new protocol using RNAscope to detect multiple mRNAs in Drosophila brains, improving signal and specificity.

## Contribution

A novel protocol for multiplexed mRNA detection in whole Drosophila brains using RNAscope and immunohistochemistry.

## Key findings

- RNAscope overcomes traditional in situ hybridization limitations like weak signal and high background.
- The protocol allows reliable quantification of mRNAs in cells targeted by Gal4/UAS systems.
- Steps for tissue preparation, transcript labeling, and quantitative analysis are detailed.

## Abstract

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

•Protocol for multiplexed RNAscope and IHC labeling in whole-mount Drosophila brains•Steps for tissue preparation, transcript labeling, and IHC•Instructions for quantitative analysis of transcript levels in labeled cells

Protocol for multiplexed RNAscope and IHC labeling in whole-mount Drosophila brains

Steps for tissue preparation, transcript labeling, and IHC

Instructions for quantitative analysis of transcript levels in labeled cells

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.