In Situ Hybridization: Fruit Fly Embryos and Tissues

Ronit Wilk1, Jack Hu1, Henry M. Krause2

1 The Donnelly Centre, University of Toronto, Toronto, Ontario, 2 Department of Molecular Genetics, University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 9.3
DOI:  10.1002/cpet.14
Online Posting Date:  November, 2017
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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 (fruit fly) embryos or tissues. Probe‐detection methods include a relatively simple alkaline phosphatase reaction, as well as higher‐resolution and higher‐throughput versions using fluorescence‐conjugated tyramide labeling. New modifications that enhance probe penetration and detection in various tissues are also provided. © 2017 by John Wiley & Sons, Inc.

Keywords: Drosophila; FISH; fluorescence; fruit fly; in situ hybridization; mRNA localization; sub‐cellular localization; tyramide; tissues

     
 
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Table of Contents

  • Overview and Principles
  • Strategic Planning
  • 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: In Situ 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
  • 10× DIG‐NTP labeling mix (see Table 9.3.2 to make the mix)
  • DIG‐UTP (digoxigenin‐11‐uridine‐5′‐triphosphate; Roche, cat. no. 11‐209‐256‐910)
  • 5× transcription buffer (supplied with RNA polymerase)
  • 40 U/μl RiboLock RNase Inhibitor (Thermo Scientific, cat. no. EO0381)
  • T3, T7, or SP6 RNA polymerase (Thermo Scientific, cat. no. EP0101, EP0111, or EP0131, respectively)
  • Diethylpyrocarbonate (DEPC)–treated water (see recipe in unit 5.2 or unit 8.2)
  • RNase‐free glycogen (Fermentas, cat. no. R0551)
  • 3 M sodium acetate, pH 5.2 (unit 3.1)
  • 70% and 100% ethanol, cold (RNase‐free)
  • 1% agarose gel made with DEPC‐treated water (unit 7.2)
  • RNA hybridization solution (see recipe)
Table 9.3.2   Materials10× DIG‐NTP Mix

Component Stock concentration Volume Final concentration
ATP 100 mM 7 μl 10 mM
CTP 100 mM 7 μl 10 mM
GTP 100 mM 7 μl 10 mM
UTP 100 mM 4.5 μl 6.5 mM
Dig‐11‐UTP 10 mM 25 μl 3.5 mM
RNase‐free water 19.5 μl
Total 70 μl

  • 0.5 or 1.5‐ml microcentrifuge tube(s) (RNase/DNase‐free) or 96‐well plates
  • RNase/DNase‐free pipet tips
  • 37°C incubator
  • Microcentrifuge
  • Additional reagents and equipment for agarose gel electrophoresis (unit 7.2)

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)
  • Fresh 10× (40%) formaldehyde solution (see recipe)
  • Fresh 4% formaldehyde solution in PBT (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: 100‐ and 800‐μm mesh (FlyStuff, cat no. 57‐103 and SEFAR–Nitex, cat. no 06‐780/53; respectively)
  • 20‐ml glass scintillation vials or glass bottles with water‐tight lids
  • 15‐ and 50‐ml conical tubes (Falcon) or 0.5‐ or 1.5‐ml microcentrifuge RNase‐free tubes
  • 96‐well microtiter plates, optional
  • RNase‐free tips
  • Wide‐mouth or cut tips to transfer embryos
  • Rocking platform
  • Mechanical shaker

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

  Additional Materials (also see protocol 2)
  • Well‐fed larvae or adult flies (or any specimen that will be dissected)
  • 4% fixing solution plus picric acid (see recipe under “Formaldehyde solutions” in Reagents and Solutions)
  • PBTT (see recipe)
  • 0.3% H 2O 2 in PBS, made fresh
  • 80% acetone, –20°C
  • PBTT plus 4% formaldehyde (see recipe under “Formaldehyde solutions” in Reagents and Solutions)
  • PBTT plus 4% formaldehyde and picric acid (see recipe under “Formaldehyde solutions” in Reagents and Solutions)
  • 9‐cm diameter Petri dish
  • Dissecting microscope and tools
  • Rocking platform

Basic Protocol 3: In Situ Hybridization

  Materials
  • RNA hybridization solution (see recipe)
  • Embryos or tissues fixed and ready for hybridization
  • DIG‐labeled RNA probes (see protocol 1)
  • PBT solution (see recipe)
  • PBTT (see recipe)
  • Boiling heating block or water bath
  • 56°C stable sand or metal beads in heating unit

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

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

Alternate Protocol 2: Probe Detection Using TSA

  Additional Materials (also see protocol 5)
  • PBTTB (see recipe)
  • Hybridized embryos or tissues ( protocol 4)
  • Anti‐DIG antibody: biotin‐conjugated mouse monoclonal anti‐DIG (use 1/400 dilution from a 1 mg/ml stock solution in PBTB; Jackson Immuno‐Research Laboratories, cat. no. 200‐062‐156)
  • PBTT (see recipe)
  • Streptavidin‐HRP conjugate (use 1/1000 dilution from a 1 mg/ml stock solution in PBTB; Molecular Probes, cat. no. S991)
  • 100× DAPI (4′,6‐diamidino‐2‐phenylindole) solution; 0.1 mg/ml; Sigma, cat. no. D‐9542)
  • Tyramide fluorescent conjugate; one of the following:
  • Cy3 tyramide conjugate (appropriate dilution of stock solution in activation buffer; Perkin Elmer Life Sciences, cat. no. SAT704A)
  • Alexa Fluor 488 tyramide conjugate (appropriate dilution of stock solution in activation buffer; Molecular Probes, cat. no. T‐20932)
  • Tyramide conjugate produced in‐house according to the published protocol of Zhou & Vize ( ), followed by appropriate dilution in activation buffer (1/5000 of a 30% stock of H 2O 2 in PBT: final concentration 0.006% H 2O 2)
  • Tyramide activation buffer (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • Antifade mounting solution (see recipe)
  • Rocking platform
IMPORTANT NOTE: If the signal at the end of the protocol is extremely high, replace the anti‐DIG biotin‐conjugated antibody and the streptavidin‐HRP conjugate with a single anti‐DIG HRP conjugated antibody “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”. Incubations and washes are the same as described in this protocol (see Figs.  , , and ).

Basic Protocol 5: Mounting Samples and Microscopy

  Materials
  • Embryo or tissue samples
  • Anti‐fade mounting solution (see recipe)
  • Clear nail polish
  • Wide‐mouth or cut pipet tips to transfer embryos or tissues
  • 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., & van Duijn, P. (1980). A new method for fluorescence microscopical localization of specific DNA sequences by in situ hybridization of fluorochrome‐labelled RNA. Experimental Cell Research, 128, 485–490. doi: 10.1016/0014‐4827(80)90087‐7.
  Buongiorno‐Nardelli, M., & Amaldi, F. (1970). Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections. Nature, 225, 946–948. doi: 10.1038/225946a0.
  Campos‐Ortega, J. A., & Hartenstein, V. (1997). The embryonic development of Drosophila melanogaster (2nd edn.). Germany: Springer.
  John, H. A., Birnstiel, M. L., & Jones, K. W. (1969). RNA‐DNA hybrids at the cytological level. Nature, 223, 582–587. doi: 10.1038/223582a0.
  Kaku, T., Ekem, J. K., Lindayen, C., Bailey, D. J., Van Nostrand, A. W., & Farber, E. (1983). Comparison of formalin‐ and acetone‐fixation for immunohistochemical detection of carcinoembryonic antigen (CEA) and keratin. American Journal of Clinical Pathology, 80, 806–815. doi: 10.1093/ajcp/80.6.806.
  Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis, W., & Bier, E. (2004). Multiplex detection of RNA expression in Drosophila embryos. Science, 305, 846. doi: 10.1126/science.1099247.
  Lecuyer, E., Parthasarathy, N., & Krause, H. M. (2008). Fluorescent in situ hybridization protocols in Drosophila embryos and tissues. Methods in Molecular Biology, 420, 289–302. doi: 10.1007/978‐1‐59745‐583‐1_18.
  Lecuyer, E., Yoshida, H., Parthasarathy, N., Alm, C., Babak, T., Cerovina, T., … Krause, H. M. (2007). Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell, 131, 174–187. doi: 10.1016/j.cell.2007.08.003.
  Levsky, J. M., & Singer, R. H. (2003). Fluorescence in situ hybridization: Past, present and future. Journal of Cell Science, 116, 2833–2838. doi: 10.1242/jcs.00633.
  Morrison, L. E., Ramakrishnan, R., Ruffalo, T. M., & Wilber, K. A. (2002). Labeling fluorescence in situ hybridization probes for genomic targets. Methods in Molecular Biology, 204, 21–40.
  Raap, A. K., van de Corput, M. P., Vervenne, R. A., van Gijlswijk, R. P., Tanke, H. J., & Wiegant, J. (1995). Ultra‐sensitive FISH using peroxidase‐mediated deposition of biotin‐ or fluorochrome tyramides. Human Molecular Genetics, 4, 529–534. doi: 10.1093/hmg/4.4.529.
  Wilk, R., Hu, J., Blotsky, D., & Krause, H. M. (2016). Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs. Genes & Development, 30, 594–609. doi: 10.1101/gad.276931.115.
  Zhou, X., & Vize, P. D. (2004). Proximo‐distal specialization of epithelial transport processes within the Xenopus pronephric kidney tubules. Developmental Biology, 271, 322–338. doi: 10.1016/j.ydbio.2004.03.036.
Internet Resources
  https://www.sdbonline.org/sites/fly/atlas/00atlas.htm
  Atlas of Drosophila Development. Hartenstein, V. 1993. Published by Cold Spring Harbor Laboratory Press.FlyBase—A Database of Drosophila Genes & Genomes.
  http://fly‐fish.ccbr.utoronto.ca/
  FlyFISH database for in situ hybridization with tyramide amplification—Krause Lab, University of Toronto.
  https://insitu.fruitfly.org
  Berkeley Drosophila Genome Project—in situ homepage ‐ Patterns of gene expression in Drosophila embryogenesis.
  https://www.fruitfly.org/about/methods/RNAinsitu.html
  Berkeley Drosophila Genome Project—BDGP Resources—96‐well RNA in situ hybridization protocol.
  https://shop.roche.com/wcsstore/RASCatalogAssetStore/Articles/05353122001_08.08.pdf
  Nonradioactive in situ hybridization application manual—Roche Diagnostics.
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