Abstract
We describe a fluorescence in situ hybridization method that permits detection of the localization and abundance of single mRNAs (smFISH) in cleared whole-mount adult Drosophila brains. The approach is rapid and multiplexable and does not require molecular amplification; it allows facile quantification of mRNA expression with subcellular resolution on a standard confocal microscope. We further demonstrate single-mRNA detection across the entire brain using a custom Bessel beam structured illumination microscope (BB-SIM).
Highlights
Brains labeled with fluorescent in situ hybridization (FISH) probes targeting Green Fluorescent Protein (GFP) and pigment dispersing factor (PDF) mRNAs in distinct colors and imaged on a confocal microscope revealed the characteristic two groups of l-LNvs and s-LNvs on each side of the brain
We acquired confocal images of light-dark cycle entrained wild type files at zeitgeber times (ZT) 2 and 14, and we observed as expected a strong Tim signal in PDF neurons at ZT14, while the intensity was greatly diminished at ZT2 (Figure 1c, 1d and S2)[9,12]
We selected three Gal[4] lines (Mi1, Mi4, Mi9)[13] with known neurotransmitter expression and crossed them with UAS-myr::HaloTag to generate expression of a HaloTag reporter in the desired neurons[14]. We profiled those brains by labeling the HaloTag protein with its fluorescent ligand in a first color (Methods) and hybridizing FISH probes targeting genes involved in distinct neurotransmitter pathways in a second color (Gad[1], vGlut and Chat, respectively associated with GABAergic, glutamatergic and cholinergic transmission)
Summary
GFP-coding mRNA signal was not observed outside of the PDF neurons or in wild type flies, confirming specificity of the hybridization (Figure 1b). To assess the impact of tissue depth on our detection performance, we measured the distribution of fluorescence intensities and spot sizes of Tim mRNAs imaged in different brain regions.
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