Abstract
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.
Highlights
In situ hybridization has been a mainstay of cell and developmental biology for determining where and when genes are expressed in wild-type or mutant cells and tissues
While single molecule fluorescent in situ hybridization (smFISH) has been used successfully in Drosophila oocytes and embryos [9, 10], only traditional RNA fluorescent in situ hybridization (FISH) methods have been used in the neuromuscular junction (NMJ) [11–13]
The relatively mild hybridization and wash conditions required for smFISH allow tissue morphology to be well preserved for meaningful biological interpretations
Summary
In situ hybridization has been a mainstay of cell and developmental biology for determining where and when genes are expressed in wild-type or mutant cells and tissues. The recent development of single molecule fluorescent in situ hybridization (smFISH) methods have increased the sensitivity, ease of application of FISH methodology, and enabled multiplexing with antibodies against. The study of RNA biology in neuroscience has been held back by the lack of suitable methods for high quality in situ hybridization in some key experimental models and tissues. While smFISH has been used successfully in Drosophila oocytes and embryos [9, 10], only traditional RNA FISH methods have been used in the NMJ [11–13]. Such methods have not been widely adopted due to variability, poor signal–noise ratios, and limited sensitivity for sparse transcript expression. We managed the relatively large number and size of image files with OMERO and created summary figures with OMERO-Figure, a platform that enables public distribution of the raw image files
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