Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction
The lack of an effective, simple, and highly sensitive protocol for fluorescent in situ hybridization (FISH) at the Drosophila larval neuromuscular junction (NMJ) has hampered the study of mRNA biology. Here, we describe our modified single molecule FISH (smFISH) methods that work well in whole mount Drosophila NMJ preparations to quantify primary transcription and count individual cytoplasmic mRNA molecules in specimens while maintaining ultrastructural preservation. The smFISH method is suitable for high-throughput sample processing and 3D image acquisition using any conventional microscopy imaging modality and is compatible with the use of antibody colabeling and transgenic fluorescent protein tags in axons, glia, synapses, and muscle cells. These attributes make the method particularly amenable to super-resolution imaging. With 3D Structured Illumination Microscopy (3D-SIM), which increases spatial resolution by a factor of 2 in X, Y, and Z, we acquire super-resolution information about the distribution of single molecules of mRNA in relation to covisualized synaptic and cellular structures. Finally, we demonstrate the use of commercial and open source software for the quality control of single transcript expression analysis, 3D-SIM data acquisition and reconstruction as well as image archiving management and presentation. Our methods now allow the detailed mechanistic and functional analysis of sparse as well as abundant mRNAs at the NMJ in their appropriate cellular context.
Key wordssmFISH Single molecule fluorescence in situ hybridization Structured Illumination Super-resolution imaging 3D-SIM Drosophila melanogaster Larval neuromuscular junction mRNA localization Synapse
We thank Talila Volk (Weizmann Institute of Science, Rehovot, Israel) for the Msp300 antibody; Flybase and the Bloomington Drosophila Stock Center for resources. We also thank David Ish-Horowicz and members of the Davis lab for discussions and comments on the manuscript. This work was supported by a Wellcome Trust Senior Basic Biomedical Research Fellowship (096144) to I.D., Wellcome Trust Strategic Awards (091911 and 107457/Z/15/Z) supporting advanced microscopy at Micron Oxford (http://micronoxford.com), and a Clarendon scholarship to LY.
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