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High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification.
| Content Provider | Europe PMC |
|---|---|
| Author | Jandura, Allison Hu, Jack Wilk, Ronit Krause, Henry M. |
| Copyright Year | 2017 |
| Abstract | In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time. |
| Journal | Journal of Visualized Experiments : JoVE |
| PubMed Central reference number | PMC5752429 |
| Issue Number | 128 |
| PubMed reference number | 29155736 |
| e-ISSN | 1940087X |
| DOI | 10.3791/56281 |
| Language | English |
| Publisher | MyJove Corporation |
| Publisher Date | 2017-10-19 |
| Access Restriction | Open |
| Rights License | This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. To view a copy of this license, visithttp://creativecommons.org/licenses/by-nc-nd/3.0/ Copyright © 2017, Journal of Visualized Experiments |
| Subject Keyword | Genetics Issue 128 RNA subcellular localization fluorescent in situ hybridization FISH Drosophila high-throughput tyramide signal amplification embryo tissues. |
| Content Type | Text |
| Resource Type | Article |
| Subject | Immunology and Microbiology Chemical Engineering Neuroscience Biochemistry, Genetics and Molecular Biology |
