Abstract
In situ hybridization (ISH) is a powerful technique for detecting nucleic acids in cells and tissues. Here we describe three ISH procedures that are optimized for Drosophila ovaries: whole-mount, digoxigenin-labeled RNA ISH; RNA fluorescent ISH (FISH); and protein immunofluorescence (IF)–RNA FISH double labeling (IF/FISH). Each procedure balances conflicting requirements for permeabilization, fixation and preservation of antigenicity to detect RNA and protein expression with high resolution and sensitivity. The ISH protocol uses alkaline phosphatase–conjugated digoxigenin antibodies followed by a color reaction, whereas FISH detection involves tyramide signal amplification (TSA). To simultaneously preserve antigens for protein detection and enable RNA probe penetration for IF/FISH, we perform IF before FISH and use xylenes and detergents to permeabilize the tissue rather than proteinase K, which can damage the antigens. ISH and FISH take 3 d to perform, whereas IF/FISH takes 5 d. Probe generation takes 1 or 2 d to perform.
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Acknowledgements
We thank G. Martin at the University of Washington Keck Center for imaging and advice on methods used in this protocol, P. Louie and N. Thayer for contributing to in situ hybridization optimization, T. Dodgen for a troubleshooting gel, J. Parrish and J. Lee for the use of video equipment to film ovary dissections, the Bloomington Stock Center for fly stocks and the Drosophila Genome Resources Center for cDNA clones. The α-Spectrin and E-cadherin monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, which was developed under the auspices of the National Institute for Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences. This work was supported by the University of Washington Provost Bridge Funds and NIH grant no. R01-GM079433 (C.A.B.), NIH/National Human Genome Research Institute grant no. T32 H600035 'Interdisciplinary Training in Genomic Sciences' (S.G.Z.) and National Science Foundation Graduate Research Fellowship no. DGE-0718124 (N.C.P.).
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C.A.B. supervised the project. C.A.B., N.C.P., A.E.A. and S.G.Z. designed the experiments. N.C.P., A.E.A. and S.G.Z. performed the experiments. C.A.B. and A.E.A. optimized the ISH methods; N.C.P., A.E.A. and S.G.Z. optimized the FISH methods; and S.G.Z. optimized the dual IF/FISH methods. N.C.P. performed dissections for Supplementary Video 1. S.G.Z. filmed and edited Supplementary Video 1. C.A.B. and S.G.Z. wrote the paper and N.C.P. and A.E.A. commented on drafts of the manuscript.
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Supplementary Video 1
Drosophila Ovary Dissection. Supplementary video showing an example of the ovary dissection procedure, including preparation, a typical dissection, dissection variations and tool maintenance. (MOV 53260 kb)
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Zimmerman, S., Peters, N., Altaras, A. et al. Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries. Nat Protoc 8, 2158–2179 (2013). https://doi.org/10.1038/nprot.2013.136
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DOI: https://doi.org/10.1038/nprot.2013.136
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