In Situ Hybridization: Fruit Fly Embryos and Tissues

Ronit Wilk1, Sreenivasa U.M. Murthy1, Haixu Yan1, Henry M. Krause1

1 The Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario, Canada
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 9.3
DOI:  10.1002/9780470089941.et0903s04
Online Posting Date:  December, 2010
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Abstract

It is well known that transcript localization controls important biological processes, including cell fate determination, cell polarity, cell migration, morphogenesis, neuronal function, and embryonic axis specification. Thus, the sub‐cellular visualization of transcripts in ‘their original place’ (in situ) is an important tool to infer and understand their trafficking, stability, translation, and biological functions. This has been made possible through the use of labeled ‘anti‐sense’ probes that can be readily detected after hybridization to their ‘sense’ counterparts. The following is a series of protocols for conducting in situ hybridization in Drosophila embryos or tissues. These methods include standard alkaline phosphatase methods, as well as higher resolution and throughput variations using fluorescence‐based probe detection. New modifications that enhance probe penetration and detection in various tissues are also provided. Curr. Protoc. Essential Lab. Tech. 4:9.3.1‐9.3.24. © 2010 by John Wiley & Sons, Inc.

Keywords: in situ hybridization; Drosophila; embryos; Drosophila; larvae; mRNA localization; sub‐cellular localization; fluorescence; tyramide; FISH

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Protocols
  • Basic Protocol 1: RNA Probe Production
  • Basic Protocol 2: Collection, Fixation, and Preparation of Embryos
  • Alternate Protocol 1: Dissection, Fixation, and Preparation of Tissues
  • Basic Protocol 3: Hybridization
  • Basic Protocol 4: Probe Detection Using Alkaline Phosphatase (AP)
  • Alternate Protocol 2: Probe Detection Using TSA
  • Basic Protocol 5: Mounting Samples and Microscopy
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: RNA Probe Production

  Materials
  • Template DNA
  • DIG‐RNA labeling mix (Roche Applied Science, cat. no. 11 277 073 910)
  • 10× transcription buffer (supplied with polymerase or 0.4 M Tris⋅Cl, pH 8.0; 60 mM MgCl 2; 100 mM dithiothreitol; 20 mM spermidine)
  • RNAguard RNase inhibitor (Amersham Biosciences, cat. no. 27 0816 01)
  • T3, T7, or SP6 RNA polymerase (Fermentas Life Sciences, cat. no. EP0101, EP0111, or EP0131, respectively)
  • Diethylpyrocarbonate (DEPC)‐treated water (unit 5.2; autoclaved)
  • 1% agarose gel
  • RNase‐free glycogen (Fermentas, cat. no. R0551)
  • 3 M sodium acetate, pH 5.2 (unit 3.3)
  • 70% and 100% ethanol, cold
  • 0.5‐ or 1.5‐ml microcentrifuge tube(s) (RNase/DNase‐free)
  • RNase/DNase‐free tips
  • 37°C incubator
  • Agarose gel electrophoresis equipment
  • Microcentrifuge

Basic Protocol 2: Collection, Fixation, and Preparation of Embryos

  Materials
  • Drosophila flies contained in cylinders/cages
  • Agar‐fruit juice plates of appropriate size (see recipe)
  • 1:1 chlorine bleach solution diluted in water (final concentration of ∼3% sodium hypochlorite; prepare fresh)
  • Heptane (HPLC‐grade; do not use commercial grade or old bottles)
  • 10× (40%) formaldehyde solution (see recipe)
  • 1× PBS solution (see recipe)
  • Methanol (HPLC‐grade)
  • PBT solution (see recipe)
  • 20 mg/ml proteinase K stock solution (see recipe)
  • 2 mg/ml glycine solution (see recipe)
  • RNA hybridization solution (see recipe)
  • Soft brushes
  • Collecting meshes/screens: 250‐ and 150‐µm mesh (60 and 100 Mesh, Fisher Scientific, respectively)
  • 20‐ml glass scintillation vials or glass bottles with water‐tight lids
  • Mechanical shaker
  • 15‐ and 50‐ml conical tubes (Falcon) or 0.5‐ or 1.5‐ml microcentrifuge RNase‐free tubes
  • 96‐well microtiter plates, optional
  • Wide‐mouth or cut tips to transfer embryos
  • Nutator mixer or rocking platform
  • RNase‐free tips

Alternate Protocol 1: Dissection, Fixation, and Preparation of Tissues

  • Well‐fed larvae
  • PBTT plus 4% formaldehyde (from fresh 40% formaldehyde stock; see recipe)
  • PBTT (see recipe)
  • 3% H 2O 2 in PBS, made fresh
  • Acetone, –20°C
  • Dissecting microscope and tools

Basic Protocol 3: Hybridization

  Materials
  • RNA hybridization solution (see recipe)
  • Embryos or tissues
  • DIG‐labeled RNA probes (see protocol 2)
  • PBT (see recipe)
  • Boiling heating block or water bath
  • 56°C stable sand bath, water bath, or oven

Basic Protocol 4: Probe Detection Using Alkaline Phosphatase (AP)

  Materials
  • PBTB (see recipe)
  • Embryos or tissues in PBT
  • Anti‐digoxigenin‐AP Fab fragments (Roche Applied Science), store at 4°C
  • Alkaline phosphatase buffer (AP buffer; see recipe)
  • PBT (see recipe)
  • AP developing solution (see recipe)
  • 100% ethanol
  • Rocking platform

Alternate Protocol 2: Probe Detection Using TSA

  • Anti‐DIG antibody:
    • For embryos: biotin‐conjugated mouse monoclonal anti‐DIG (1/400 dilution of a 1 mg/ml stock solution in PBTB; Jackson ImmunoResearch Laboratories, cat. no. 200‐062‐156) or
    • For tissues: HRP‐conjugated mouse monoclonal anti‐DIG (1/500 dilution of a 1 mg/ml stock solution in PBTB (Jackson Immuno‐Research Laboratories, cat. no. 200‐032‐156)
  • 100× DAPI (4′,6‐diamidino‐2‐phenylindole) solution (0.1 mg/ml; Sigma, cat. no. D‐9542)
  • Streptavidin‐HRP conjugate (1/1000 dilution from a 1 mg/ml stock solution in PBTB (Molecular Probes, cat. no. S991) (for embryos)
  • Fluorescent conjugate:
    • Cy3 tyramide conjugate (1/50 dilution of stock solution in amplification buffer; Perkin Elmer Life Sciences, cat. no. SAT704A) or
    • Alexa Fluor 488 tyramide conjugate (1/50 dilution of stock solution in amplification buffer; Molecular Probes, cat. no. T‐20932)

Basic Protocol 5: Mounting Samples and Microscopy

  Materials
  • Embryo or tissue samples
  • Anti‐fade mounting solution (see recipe) or 70% glycerol
  • Clear nail polish
  • Wide‐mouth or cut tips to transfer embryos or tissue
  • Microscope slides
  • Microscope coverslips (22 × 22–mm)
  • Fluorescence or confocal microscope equipped with all the required filters and a sensitive digital camera
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Figures

Videos

Literature Cited

   Bauman, J.G., Wiegant, J., Borst, P., and van Duijn, P. 1980. A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome labeled RNA. Exp. Cell Res. 128:485‐490.
   Brady, M.A.W. and Finlan, M.F. 1990. Radioactive labels: Autoradiography and choice of emulsions for in situ hybridization. In In Situ Hybridization: Principle and Practice (J.M. Polak and J. O'D. McGee, eds.) Oxford University Press, Oxford.
   Buongiorno‐Nardelli, M. and Amaldi, F. 1969. Autoradiographic detection of molecular hybrids between rRNA and DNA in tissue sections. Nature 225:946‐947.
   Campos‐Ortega, J.A. and Hartenstein, V. 1997. The Embryonic Development of Drosophila melanogaster. Springer‐Verlag, Germany.
   Hartenstein, V. 1993. Atlas of Drosophila Development. Cold Spring Harbor Laboratory Press, New York. (http://www.sdbonline.org/fly/atlas/00atlas.htm)
   John, H.L., Birnstiel, M.L., and Jones, K.W. 1969. RNA‐DNA hybrids at the cytological level. Nature 223:912‐913.
   Kaku, T., Ekem, J.K., Lindayen, C., Bailey, D.J., Van Nostrand, A.W., and Farber, E. 1983. Comparison of formalin‐ and acetone‐fixation for immunohistochemical detection of carcinoembryonic antigen (CEA) and keratin. Am. J. Clin. Pathol. 80:806‐815.
   Kosman, D., Mizutani, C.M., Lemons, D., Cox, W.G., McGinnis, W., and Bier, E. 2004. Multiplex detection of RNA expression in Drosophila embryos. Science 305:846.
   Lécuyer, E., Yoshida, H., Parthasarathy, N., Alm, C., Babak, T., Cerovina, T., Hughes, T.R., Tomancak, P., and Krause, H.M. 2007. Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131:174‐187.
   Lécuyer, E., Parthasarathy, N., and Krause, H.M. 2008. Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. Methods Mol. Biol. 420:289‐302.
   Levsky, J.M. and Singer, R.H. 2003. Fluorescence in situ hybridization: Past, present and future. J. Cell Sci. 116:2833‐2838.
   Morrison, L.E., Ramakrishnan, R., Ruffalo, T.M., and Wilber, K.A. 2003. Labeling fluorescence in situ hybridization probes for genomic targets. In Methods in Molecular, Vol. 204 (Y.S. Fan, ed.) Humana Press, Totowa, New Jersey.
   Nagaso, H., Murata, T., Day, N., and Yokoyama, K.K. 2001. Simultaneous detection of RNA and protein by in situ hybridization and immunological staining. J. Histochem. Cytochem. 49:1177‐1182.
   Raap, A.K., van de Corput, M.P.C., Vervenne, R.A.W., van Gijlswijk, R.P.M., Tanke, H.J., and Wiegant, J. 1995. Ultra‐sensitive FISH using peroxidase‐mediated deposition of biotin‐ or fluorochrome tyramides. Hum. Mol. Genet. 4:529‐534.
   Tomancak, P., Beaton, A., Weiszmann, R., Kwan, E., Shu, S., Lewis, S.E., Richards, S., Ashburner, M., Hartenstein, V., Celniker, S.E., and Rubin, G.M. 2002. Systematic determination of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 3:RESEARCH0088.
   Tomancak, P., Berman, B.P., Beaton, A., Weiszmann, R., Kwan, E., Hartenstein, V., Celniker, S.E., and Rubin, G.M. 2007. Global analysis of patterns of gene expression during Drosophila embryogenesis. Genome Biol. 8:R145.
Internet Resources
  http://www.fruitfly.org/about/methods/RNAinsitu.html
  Berkeley Drosophila Genome Project—BDGP Resources—96‐well RNA in situ hybridization protocol.
  http://www.sdbonline.org/fly/atlas/00atlas.htm
  Hartenstein, V. 1993. Atlas of Drosophila Development. Published by Cold Spring Harbor Laboratory Press.
  http://www.roche‐applied‐science.com/PROD_INF/MANUALS/InSitu/InSi_toc.htm
  Nonradioactive in situ hybridization application manual—Roche Diagnostics.
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