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

Regulation of Drosophila courtship behavior by the Tlx/tailless-like nuclear receptor, dissatisfaction

“…We reexamined dsf mRNA expression in the adult CNS by in situ hybridization chain reaction (HCR), a robust, quantitative, and sensitive method to detect mRNAs in wholemounted tissues. [14][15][16][17] Using HCR probe-sets directed against dsf, we observed dsf mRNAs in the Canton S CNS in substantially more cells than originally reported (Figures 1A and 1B). In the brain, dsf expression was found in several broadly distributed clusters of cells located mostly on the anterior side, dorsal and ventral to the foramen of the esophagus.…”

“…in situ HCR and immunohistochemistry Nervous systems were dissected in 1X PBS, fixed in 4% paraformaldehyde buffered in PBS for 35 min at room temperature, and rinsed and washed in PBT (PBS with 1% Triton X-100). in situ hybridization chain reaction experiments were conducted as described 17 using HCR probe-sets (dsf-B1, Lot # PRF061) and hairpins (B1 488 HCR Amplifier) synthesized by Molecular InstrumentsÔ. We used a probe-set size of 20.…”

Optimization of Whole Mount RNA Multiplexed in situ Hybridization Chain Reaction With Immunohistochemistry, Clearing and Imaging to Visualize Octopus Embryonic Neurogenesis

“…The compatibility and efficiency of HCR with CLARITY and iDISCO+ in fresh-frozen rodent brain tissues and postmortem human brain blocks was previously tested (Kumar et al, 2021). An alternative for clearing methods is fluorescence tomography or confocal imaging ( Guo et al, 2019 ; Duckhorn et al, 2021 ). For instance, combination of whole mount HCR with fluorescence tomography has been used for finding the exact location of Cre mRNA in a Thy1-Cre mouse brain ( Guo et al, 2019 ).…”

Differentiation signals from glia are fine-tuned to set neuronal numbers during development

“…To detect spi transcripts by in situ hybridization chain reaction, we dissected, fixed and permeabilized the optic lobes as above before following the protocol described in 39 . We used the following primary antibodies in this study: mouse anti-Dac [2][3]…”

“…To determine if spi, Col4a1 and Ddr transcripts were present in the xg O , we performed HCR as detailed in Duckhorn et al (2022). We designed 20-21 probe pairs against target genes, excluding regions of strong similarity to other transcripts, with corresponding initiator sequences for amplifiers B3 (Choi et al, 2018).…”