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Optimized RNA ISH, RNA FISH and protein-RNA double labeling (IF/FISH) in Drosophila ovaries

Nature Protocols volume 8, pages 2158–2179 (2013)Cite this article

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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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Figure 1: Workflow diagram for ISH, FISH and dual protein immunofluorescent staining and FISH (IF/FISH).
Figure 2: Comparison of permeabilization methods using ISH.
Figure 3: Optimization of dual protein and RNA analyses.
Figure 4: Detection of rare, moderate and abundant transcripts with ISH and FISH.
Figure 5: Optimized protein IF/FISH of Stage 10B egg chambers.
Figure 6: Probe verification and quantification.

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References

  1. Gall, J.G. & Pardue, M.L. Formation and detection of RNA–DNA hybrid molecules in cytological preparations. Proc. Natl. Acad. Sci. USA 63, 378–383 (1969).

    Article  CAS  Google Scholar 

  2. Pardue, M.L. & Gall, J.G. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. USA 64, 600–604 (1969).

    Article  CAS  Google Scholar 

  3. Tautz, D. & Pfeifle, C. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81–85 (1989).

    Article  CAS  Google Scholar 

  4. Kallioniemi, A. et al. Comparative genomic hybridization for molecular cytogenetic analysis of solid tumors. Science 258, 818–821 (1992).

    Article  CAS  Google Scholar 

  5. Melton, D.A. et al. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056 (1984).

    Article  CAS  Google Scholar 

  6. Kallioniemi, A., Visakorpi, T., Karhu, R., Pinkel, D. & Kallioniemi, O.P. Gene copy number analysis by fluorescence in situ hybridization and comparative genomic hybridization. Methods 9, 113–121 (1996).

    Article  CAS  Google Scholar 

  7. McNeil, J.A., Johnson, C.V., Carter, K.C., Singer, R.H. & Lawrence, J.B. Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization. Genet. Anal. Tech. Appl. 8, 4158 (1991).

    Google Scholar 

  8. Wiedorn, K.H., Kuhl, H., Galle, J., Caselitz, J. & Vollmer, E. Comparison of in-situ hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV. Histochem. Cell Biol. 111, 89–95 (1999).

    Article  CAS  Google Scholar 

  9. De Block, M. & Debrouwer, D. RNA-RNA in situ hybridization using digoxigenin-labeled probes: the use of high-molecular-weight polyvinyl alcohol in the alkaline phosphatase indoxyl-nitroblue tetrazolium reaction. Anal. Biochem. 215, 86–89 (1993).

    Article  CAS  Google Scholar 

  10. Harlow, E. & Lane, D. in Antibodies: A Laboratory Manual Ch. 10, 396–399 (Cold Spring Harbor Laboratory Press, 1988).

  11. O'Neill, J.W. & Bier, E. Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. Biotechniques 17, 870, 874–875 (1994).

    Google Scholar 

  12. Yakoby, N. et al. A combinatorial code for pattern formation in Drosophila oogenesis. Dev. Cell 15, 725–737 (2008).

    Article  CAS  Google Scholar 

  13. Lécuyer, E., Parthasarathy, N. & Krause,, H.M. Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. in Drosophila: Methods and Protocols, Methods in Molecular Biology (ed. Dahmann, C.) Ch. 18, 289–302 (Humana Press, 2008).

  14. Wilkie, G.S., Shermoen, A.W., O'Farrell, P.H. & Davis, I. Transcribed genes are localized according to chromosomal position within polarized Drosophila embryonic nuclei. Curr. Biol. 9, 1263–1266 (1999).

    Article  CAS  Google Scholar 

  15. Fuchs, A., Cheung, L.S., Charbonnier, E., Shvartsman, S.Y. & Pyrowolakis, G. Transcriptional interpretation of the EGF receptor signaling gradient. Proc. Natl. Acad. Sci. USA 109, 1572–1577 (2012).

    Article  CAS  Google Scholar 

  16. Kosman, D. et al. Multiplex detection of RNA expression in Drosophila embryos. Science 305, 846 (2004).

    Article  CAS  Google Scholar 

  17. van Gijlswijk, R.P. et al. Fluorochrome-labeled tyramides: use in immunocytochemistry and fluorescence in situ hybridization. J. Histochem. Cytochem. 45, 375–382 (1997).

    Article  CAS  Google Scholar 

  18. Spradling, A.C. Developmental genetics of oogenesis. in The Development of Drosophila melanogaster, Vol. I (eds. Bate, M. & Martinez Arias, A.) 1–70 (Cold Spring Harbor Laboratory Press, 1993).

  19. Weiszmann, R., Hammonds, A.S. & Celniker, S.E. Determination of gene expression patterns using high-throughput RNA in situ hybridization to whole-mount Drosophila embryos. Nat. Protoc. 4, 605–618 (2009).

    Article  CAS  Google Scholar 

  20. Nagaso, H., Murata, T., Day, N. & Yokoyama, K.K. Simultaneous detection of RNA and protein by in situ hybridization and immunological staining. J. Histochem. Cytochem. 49, 1177–1182 (2001).

    Article  CAS  Google Scholar 

  21. Morris, C.A., Benson, E. & White-Cooper, H. Determination of gene expression patterns using in situ hybridization to Drosophila testes. Nat. Protoc. 4, 1807–1819 (2009).

    Article  CAS  Google Scholar 

  22. Toledano, H., D'Alterio, C., Loza-Coll, M. & Jones, D.L. Dual fluorescence detection of protein and RNA in Drosophila tissues. Nat. Protoc. 7, 1808–1817 (2012).

    Article  CAS  Google Scholar 

  23. Brivanlou, A.H. & Harland, R.M. Expression of an engrailed-related protein is induced in the anterior neural ectoderm of early Xenopus embryos. Development 106, 611–617 (1989).

    CAS  PubMed  Google Scholar 

  24. Harland, R.M. In situ hybridization: an improved whole-mount method for Xenopus embryos. Methods Cell Biol. 36, 685–695 (1991).

    Article  CAS  Google Scholar 

  25. Thisse, C. & Thisse, B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat. Protoc. 3, 59–69 (2008).

    Article  CAS  Google Scholar 

  26. Piette, D., Hendrickx, M., Willems, E., Kemp, C.R. & Leyns, L. An optimized procedure for whole-mount in situ hybridization on mouse embryos and embryoid bodies. Nat. Protoc. 3, 1194–1201 (2008).

    Article  CAS  Google Scholar 

  27. Acloque, H., Wilkinson, D.G. & Nieto, M.A. In situ hybridization analysis of chick embryos in whole-mount and tissue sections. Methods Cell Biol. 87, 169–185 (2008).

    Article  CAS  Google Scholar 

  28. Gillespie, D.E. & Berg, C.A. Homeless is required for RNA localization in Drosophila oogenesis and encodes a new member of the DE-H family of RNA-dependent ATPases. Genes Dev. 9, 2495–2508 (1995).

    Article  CAS  Google Scholar 

  29. Neuman-Silberberg, F.S. & Schupbach, T. The Drosophila dorsoventral patterning gene gurken produces a dorsally localized RNA and encodes a TGF-α–like protein. Cell 75, 165–174 (1993).

    Article  CAS  Google Scholar 

  30. Ruohola, H. et al. Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila. Cell 66, 433–449 (1991).

    Article  CAS  Google Scholar 

  31. Ephrussi, A., Dickinson, L.K. & Lehmann, R. oskar organizes the germ plasm and directs localization of the posterior determinant nanos. Cell 66, 37–50 (1991).

    Article  CAS  Google Scholar 

  32. Lehmann, R. & Tautz, D. In situ hybridization to RNA. in Methods in Cell Biology, Vol. 44, 575–598 (Elsevier, 1994).

    Article  CAS  Google Scholar 

  33. Sambrook, J. & Russell, D. Molecular Cloning: A Laboratory Manual 3rd edn., Vol. 1. (Cold Spring Harbor Laboratory Press, 2001).

  34. Schulz, C. In situ hybridization to Drosophila testes. Cold Spring Harb. Protoc. 10.1101/pdb.prot4764 (2007).

  35. Vize, P.D., McCoy, K.E. & Zhou, X. Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. Nat. Protoc. 4, 975–983 (2009).

    Article  Google Scholar 

  36. Wang, X. et al. Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary. Dev. Cell 10, 483–495 (2006).

    Article  CAS  Google Scholar 

  37. Pizette, S., Rabouille, C., Cohen, S.M. & Therond, P. Glycosphingolipids control the extracellular gradient of the Drosophila EGFR ligand Gurken. Development 136, 551–561 (2009).

    Article  CAS  Google Scholar 

  38. Kitadate, Y., Shigenobu, S., Arita, K. & Kobayashi, S. Boss/Sev signaling from germline to soma restricts germline-stem-cell-niche formation in the anterior region of Drosophila male gonads. Dev. Cell 13, 151–159 (2007).

    Article  CAS  Google Scholar 

  39. Lécuyer, E., Necakov, A.S., Caceres, L. & Krause, H.M. High-resolution fluorescent in situ hybridization of Drosophila embryos and tissues. Cold Spring Harb. Protoc. 10.1101/pdb.prot5019 (2008).

  40. Lerner, D.W. et al. A Rab10-dependent mechanism for polarized basement membrane secretion during organ morphogenesis. Dev. Cell 24, 159–168 (2013).

    Article  CAS  Google Scholar 

  41. Schotman, H., Karhinen, L. & Rabouille, C. dGRASP-mediated noncanonical integrin secretion is required for Drosophila epithelial remodeling. Dev. Cell 14, 171–182 (2008).

    Article  CAS  Google Scholar 

  42. Vanzo, N.F. & Ephrussi, A. Oskar anchoring restricts pole plasm formation to the posterior of the Drosophila oocyte. Development 129, 3705–3714 (2002).

    CAS  PubMed  Google Scholar 

  43. Hughes, S.C. & Krause, H.M. Single and double FISH protocols for Drosophila. in Confocal Microscopy: Methods and Protocols (ed. Paddock, S.W.) Ch. 5, 93–101 (Humana Press, 1999).

  44. Braissant, O. & Wahli, W. A simplified in situ hybridization protocol using non-radioactively labeled probes to detect abundant and rare mRNAs on tissue sections. Biochemica 1, 10–16 (1998).

    Google Scholar 

  45. Langer, P.R., Waldrop, A.A. & Ward, D.C. Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc. Natl. Acad. Sci. USA 78, 6633–6637 (1981).

    Article  CAS  Google Scholar 

  46. Peters, N.C., Thayer, N.H., Kerr, S.A., Tompa, M. & Berg, C.A. Following the 'tracks': Tramtrack69 regulates epithelial tube expansion in the Drosophila ovary through Paxillin, Dynamin, and the homeobox protein Mirror. Dev. Biol. 378, 154–169 (2013).

    Article  CAS  Google Scholar 

  47. Geisbrecht, E.R. et al. Genetic interaction screens identify a role for Hedgehog signaling in Drosophila border cell migration. Dev. Dyn. 242, 414–431 (2013).

    Article  CAS  Google Scholar 

  48. Kucherenko, M.M., Barth, J., Fiala, A. & Shcherbata, H.R. Steroid-induced microRNA let-7 acts as a spatio-temporal code for neuronal cell fate in the developing Drosophila brain. EMBO J. 31, 4511–4523 (2012).

    Article  CAS  Google Scholar 

  49. McLean, P.F. & Cooley, L. Protein equilibration through somatic ring canals in Drosophila. Science 340, 1445–1447 (2013).

    Article  CAS  Google Scholar 

  50. Dernburg, A.F. Hybridization to tissues in suspension for whole-mount FISH in Drosophila. Cold Spring Harb. Protoc. 2011, 1534–1537 (2011).

    Article  Google Scholar 

  51. Dernburg, A.F. Manual dissection and fixation of Drosophila egg chambers for whole-mount FISH. Cold Spring Harb. Protoc. 2011, 1531–1533 (2011).

    Article  Google Scholar 

  52. Dernburg, A.F. Fragmentation and labeling of probe DNA for whole-mount FISH in Drosophila. Cold Spring Harb. Protoc. 2011, 1527–1530 (2011).

    Article  Google Scholar 

  53. Becalska, A.N. & Gavis, E.R. Lighting up mRNA localization in Drosophila oogenesis. Development 136, 2493–2503 (2009).

    Article  CAS  Google Scholar 

  54. Forrest, K.M. & Gavis, E.R. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr. Biol. 13, 1159–1168 (2003).

    Article  CAS  Google Scholar 

  55. Randolph, J.B. & Waggoner, A.S. Stability, specificity and fluorescence brightness of multiply-labeled fluorescent DNA probes. Nucleic Acids Res. 25, 2923–2929 (1997).

    Article  CAS  Google Scholar 

  56. Zhang, D.Y., Chen, S.X. & Yin, P. Optimizing the specificity of nucleic acid hybridization. Nat. Chem. 4, 208–214 (2012).

    Article  CAS  Google Scholar 

  57. Femino, A.M., Fay, F.S., Fogarty, K. & Singer, R.H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  Google Scholar 

  58. Kislauskis, E.H., Li, Z., Singer, R.H. & Taneja, K.L. Isoform-specific 3′-untranslated sequences sort α-cardiac and β-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. J. Cell Biol. 123, 165–172 (1993).

    Article  CAS  Google Scholar 

  59. Blanco, A.M., Rausell, L., Aguado, B., Perez-Alonso, M. & Artero, R. A FRET-based assay for characterization of alternative splicing events using peptide nucleic acid fluorescence in situ hybridization. Nucleic Acids Res. 37, e116 (2009).

    Article  Google Scholar 

  60. Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497–1500 (1991).

    Article  CAS  Google Scholar 

  61. Larsson, C., Grundberg, I., Soderberg, O. & Nilsson, M. In situ detection and genotyping of individual mRNA molecules. Nat. Methods 7, 395–397 (2010).

    Article  CAS  Google Scholar 

  62. Nilsson, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science 265, 2085–2088 (1994).

    Article  CAS  Google Scholar 

  63. Lecuyer, E. et al. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131, 174–187 (2007).

    Article  CAS  Google Scholar 

  64. Simin, K. et al. Profiling patterned transcripts in Drosophila embryos. Genome Res. 12, 1040–1047 (2002).

    Article  CAS  Google Scholar 

  65. Tomancak, P. et al. Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 3, research0088.1 (2002).

    Article  Google Scholar 

  66. Tomancak, P. et al. Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 8, R145 (2007).

    Article  Google Scholar 

  67. Blackshaw, S. High-throughput RNA in situ hybridization in mouse retina. Methods Mol. Biol. 935, 215–226 (2013).

    Article  CAS  Google Scholar 

  68. Chiao, E., Leonard, J., Dickinson, K. & Baker, J.C. High-throughput functional screen of mouse gastrula cDNA libraries reveals new components of endoderm and mesoderm specification. Genome Res. 15, 44–53 (2005).

    Article  CAS  Google Scholar 

  69. Quiring, R. et al. Large-scale expression screening by automated whole-mount in situ hybridization. Mech. Dev. 121, 971–976 (2004).

    Article  CAS  Google Scholar 

  70. King, R.C. Ovarian Development in Drosophila melanogaster (Academic Press, 1970).

  71. Sive, H.L., Grainger, R.M. & Harland, R.M. Synthesis and purification of digoxigenin-labeled RNA probes for in situ hybridization. Cold Spring Harb. Protoc. 10.1101/pdb.prot4778 (2007).

  72. Parrish, J.Z., Kim, M.D., Jan, L.Y. & Jan, Y.N. Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites. Genes Dev. 20, 820–835 (2006).

    Article  CAS  Google Scholar 

  73. Kessler, C. Factors influencing nucleic acid hybridization. in Nonradioactive Labeling and Detection of Biomolecules (ed. Kessler, C.) Ch. 19, 253–264 (Springer, 1992).

  74. Bodkin, D.K. & Knudson, D.L. Genetic relatedness of Palyam serogroup viruses by RNA-RNA blot hybridization. J. Gen. Virol. 67 (Part 4): 683–691 (1986).

    Article  CAS  Google Scholar 

  75. Dubreuil, R., Byers, T.J., Branton, D., Goldstein, L.S. & Kiehart, D.P. Drosophilia spectrin. I. Characterization of the purified protein. J. Cell Biol. 105, 2095–2102 (1987).

    Article  CAS  Google Scholar 

  76. Kim, Y. et al. Gene regulation by MAPK substrate competition. Dev. Cell 20, 880–887 (2011).

    Article  CAS  Google Scholar 

  77. Oda, H., Uemura, T., Harada, Y., Iwai, Y. & Takeichi, M. A Drosophila homolog of Cadherin associated with Armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165, 716–726 (1994).

    Article  CAS  Google Scholar 

  78. Hauptmann, G. & Gerster, T. Multicolor whole-mount in situ hybridization. Methods Mol. Biol. 137, 139–148 (2000).

    CAS  PubMed  Google Scholar 

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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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  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Sandra G Zimmerman, Nathaniel C Peters, Ariel E Altaras & Celeste A Berg

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Contributions

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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Correspondence to Celeste A Berg.

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Supplementary information

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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