Chapter title |
Super-Resolution Single Molecule FISH at the Drosophila Neuromuscular Junction
|
---|---|
Chapter number | 10 |
Book title |
RNA Detection
|
Published in |
Methods in molecular biology, January 2018
|
DOI | 10.1007/978-1-4939-7213-5_10 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7212-8, 978-1-4939-7213-5
|
Authors |
Joshua S. Titlow, Lu Yang, Richard M. Parton, Ana Palanca, Ilan Davis |
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. |
Twitter Demographics
Geographical breakdown
Country | Count | As % |
---|---|---|
United Kingdom | 9 | 27% |
United States | 6 | 18% |
France | 2 | 6% |
Switzerland | 1 | 3% |
Canada | 1 | 3% |
Unknown | 14 | 42% |
Demographic breakdown
Type | Count | As % |
---|---|---|
Members of the public | 23 | 70% |
Scientists | 9 | 27% |
Science communicators (journalists, bloggers, editors) | 1 | 3% |
Mendeley readers
Geographical breakdown
Country | Count | As % |
---|---|---|
Unknown | 28 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
---|---|---|
Student > Bachelor | 6 | 21% |
Student > Master | 4 | 14% |
Unspecified | 3 | 11% |
Professor > Associate Professor | 2 | 7% |
Student > Ph. D. Student | 2 | 7% |
Other | 5 | 18% |
Unknown | 6 | 21% |
Readers by discipline | Count | As % |
---|---|---|
Biochemistry, Genetics and Molecular Biology | 8 | 29% |
Agricultural and Biological Sciences | 6 | 21% |
Unspecified | 3 | 11% |
Neuroscience | 2 | 7% |
Medicine and Dentistry | 1 | 4% |
Other | 1 | 4% |
Unknown | 7 | 25% |