In Situ Hybridization and Detection Using Nonisotopic Probes

Joan H.M. Knoll1, Peter Lichter2, Khldoun Bakdounes3, Isam‐Eldin A. Eltoum3

1 University of Western Ontario, London, 2 Deutsches Krebsforschungszentrum, Heidelberg, 3 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.7
DOI:  10.1002/0471142727.mb1407s79
Online Posting Date:  July, 2007
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Nonisotopic in situ hybridization can be used to determine the cellular location and relative levels of expression for specific transcripts within cells and tissues. RNA in specimen preparations is hybridized with a biotin‐ or digoxigenin‐labeled probe, which is generally detected by fluorescence or enzymatic methods. Fluorescence in situ hybridization (FISH), probably the most widely used method, is described here, along with amplification of weak FISH signals. Nonisotopic probes can also be detected by enzymatic reactions using horseradish peroxidase or alkaline phosphatase, as described here. Curr. Protoc. Mol. Biol. 79:14.7.1‐14.7.17. © 2007 by John Wiley & Sons, Inc.

Keywords: nonisotopic detection; in situ hybridization; FISH; enzymatic detection

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Table of Contents

  • Introduction
  • Basic Protocol 1: Fluorescence in Situ Hybridization
  • Support Protocol 1: Amplification of Signals from Biotinylated Probes
  • Support Protocol 2: Amplification of Signals from Digoxigenin‐Labeled Probes
  • Basic Protocol 2: Enzymatic Detection Using Horseradish Peroxidase
  • Alternate Protocol 1: Enzymatic Detection Using Alkaline Phosphatase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Fluorescence in Situ Hybridization

  • Specimen: paraffin sections (unit 14.1) or cryosections (unit 14.2) on microscope slides
  • 100 to 150 ng nonisotopically labeled DNA probe (unit 3.18)
  • Deionized formamide (American Bioanalytical or Sigma‐Aldrich, or prepare as in unit 14.3)
  • 10 mg/ml sonicated salmon sperm DNA (see recipe)
  • Master hybridization mix (see recipe)
  • 50% (v/v) formamide/2× SSC (deionized formamide not necessary)
  • 20× SSC, pH 7.0 ( appendix 22)
  • Biotin, digoxigenin, or biotin/digoxigenin detection solution (see reciperecipes)
  • 0.1% (v/v) Triton X‐100/4× SSC
  • DAPI or propidium iodide staining solution (see reciperecipes)
  • Appropriate antifade mounting medium (see recipe)
  • Clear nail polish
  • 37° and 70° to 80°C water baths
  • 22 × 22–mm coverslips
  • Moist chamber (e.g., Fig. )
  • Rubber cement (for overnight hybridization)
  • Coplin jars
  • Aluminum foil
  • Slide box with desiccant (VWR Scientific)
  • Fluorescence microscope with epi‐illumination and filter sets (Chroma Technology or Omega Optical) appropriate for fluorochromes used: e.g., single‐band‐pass filters (fluorescein, Texas red, rhodamine, DAPI, Quantum or Q‐dots), dual‐band‐pass filter (fluorescein/Texas red), or triple‐band‐pass filter (fluorescein/Texas red/DAPI)
  • Computerized microscope imaging system (e.g., Applied Imaging) or 35‐mm camera with standard photography film (Kodak Ektar‐1000 or Ektachrome‐400 color film or Technical Pan 2415 black‐and‐white film)
  • Additional reagents and equipment for preparing paraffin sections, cryosections, or cells for hybridization (unit 14.3), and for ethanol precipitation of DNA (unit 2.1)
CAUTION: Formamide, DAPI, and propidium iodide are hazardous; see manufacturer's information for guidelines on handling, storage, and disposal.

Support Protocol 1: Amplification of Signals from Biotinylated Probes

  • 1 to 3 µg/ml biotinylated anti‐avidin antibody (Vector Laboratories) in 4× SSC/1% (w/v) BSA (fraction V)
  • 2 to 5 µg/ml fluorescein‐avidin DCS (Vector Laboratories) in 4× SSC/1% (w/v) BSA (fraction V)

Support Protocol 2: Amplification of Signals from Digoxigenin‐Labeled Probes

  • 10 µg/ml Fab fragment of sheep anti‐digoxigenin (Roche Applied Science) in 4× SSC/1% (w/v) BSA (fraction V)
  • 3.5 to 7.0 µg/ml fluorescein‐conjugated rabbit anti–sheep IgG (Sigma‐Aldrich) in 4× SSC/1% (w/v) BSA (fraction V)

Basic Protocol 2: Enzymatic Detection Using Horseradish Peroxidase

  • Blocking solution: 1% (w/v) BSA in PBS ( appendix 22) or 5% nonfat milk and 0.1% (v/v) Tween 20 in 4× SSC ( appendix 22)
  • Streptavidin solution (see recipe)
  • 0.1% (v/v) Tween 20/PBS ( appendix 22), 42°C
  • Biotinylated HRP solution (see recipe)
  • Biotinylated tyramide solution and secondary streptavidin‐peroxidase (Dako; optional)
  • 3% H 2O 2
  • DAB substrate solution: 500 µg/ml 3,3′‐diaminobenzidine tetrahydrochloride (DAB) in PBS ( appendix 22), prepared fresh
  • PBS ( appendix 22)
  • 70%, 90%, and 100% ethanol
  • Xylene
  • Permanent mounting medium (e.g., Permount)
  • Moist chamber (Fig. )
  • Coplin jars
  • 42°C shaking water bath
  • 24 × 60–mm coverslips
  • Additional reagents and equipment for hybridization and washing (see protocol 1) and counterstaining with hematoxylin (unit 14.5)
CAUTION: DAB is hazardous; see manufacturer's information for guidelines on handling, storage, and disposal.

Alternate Protocol 1: Enzymatic Detection Using Alkaline Phosphatase

  • Biotinylated AP solution (see recipe)
  • Alkaline phosphatase buffer, pH 9.5 (see recipe), 42°C
  • NBT/BCIP substrate solution (see recipe)
  • Nuclear counterstain: 0.1% fast red (Sigma) in 5% (w/v) aluminum sulfate or 0.03% methyl green (Sigma) in H 2O
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Literature Cited

Literature Cited
   Adams, J.C. 1992. Biotin amplification of biotin and horseradish peroxidase signals in histochemical stains. J. Histochem. Cytochem. 40:1457‐1463.
   Cao, Y. 2002. In situ immuno‐PCR. A newly developed method for highly sensitive antigen detection in situ. Methods Mol. Biol. 193:191‐196.
   Clemson, C.M., McNeil, J.A., Willard, H.F., and Lawrence, J.B. 1996. XIST RNA paints the inactive X chromosome at interphase: Evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132:259‐275.
   Emmerich, P., Loos, P., Jauch, A., Hopman, A.H.N., Wiegant, J., Higgins, M., White, B.N., van der Ploeg, M., Cremer, C., and Cremer, T. 1989. Double in situ hybridization in combination with digitized image analysis: A new approach to study interphase chromosome topography. Exp. Cell Res. 181:126‐140.
   Hanna, W.M. and Kwok, K. 2006. Chromogenic in‐situ hybridization: A viable alternative to fluorescence in‐situ hybridization in the HER2 testing algorithm. Modern Pathol. 19:481‐487.
   Johnson, G.D. and de C. Nogueira Araujo, G.M. 1981. A simple method of reducing the fading of immunofluorescence during microscopy. J. Immunol. Methods 43:349‐350.
   Johnson, G.D., Davidson, R.S., McNamee, K.C., Russell, G., Goodwin, D., and Holborrow, E.L. 1982. Fading of immunofluorescence during microscopy: A study of its phenomenon and its remedy. J. Immunol. Methods 55:231‐242.
   Knoll, J.H.M. 2006. Human chromosome metaphase FISH using quantum dot conjugates. In Methods in Molecular Biology Series on Quantum Dots in Biology (C. Hotz and M. Bruchez, eds.) vol. 374, pp. 55‐60. Humana Press, Totowa, N.J.
   Knoll, J.H.M. and Lichter, P. 2005. In situ hybridization to metaphase chromosomes and interphase nuclei. Curr. Protoc. Hum. Genet. 45:4.3.1‐4.3.31.
   Knoll, J.H.M. and Rogan, P.K. 2003. Sequence‐based, in situ detection of chromosomal abnormalities at high resolution. Am. J. Med. Genet. A. 121:245‐257.
   Laakso, M., Tanner, M., and Isola, J. 2006. Dual‐color chromogenic in situ hybridization for testing of HER‐2 oncogene amplification in archival breast tumours. J. Pathol. 210:3‐9.
   Lambros, M.B., Simpson, P.T., Jones, C., Natrajan, R., Westbury, C., Steele, D., Savage, K., Mackay, A., Schmitt, F.C., Ashworth, A., and Reis‐Filho, J.S. 2006. Unlocking pathology archives for molecular genetic studies: A reliable method to generate probes for chromogenic and fluorescent in situ hybridization. Lab. Invest. 86:398‐408.
   Lawrence, J.B. and Singer, R.H. 1988. Quantitative analysis of in situ hybridization methods for the detection of actin gene expression. Nucl. Acids Res. 13:1777‐1799.
   Lawrence, J.B., Villnave, C.A., and Singer, R.H. 1988. Sensitive, high‐resolution chromatin and chromosome mapping in situ: Presence and orientation of two closely integrated copies of EBV in a lymphoma cell line. Cell 52:51‐61.
   Lawrence, J.B., Singer, R.H., and Marselle, L.M. 1989. Highly localized tracks of specific transcripts within interphase nuclei visualized by in situ hybridization. Cell 57:493‐502.
   Levsky, J.M. and Singer, R.H. 2003. Fluorescence in situ hybridization: Past, present and future. J. Cell Sci. 116:2833‐2838.
   Lichter, P., Cremer, T., Tang, C.C., Watkins, P.C., Manuelidis, L., and Ward, D.C. 1988. Rapid detection of human chromosome 21 aberrations by in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 85:9664‐9668.
   Lichter, P., Tang, C.C., Call, K., Hermanson, G., Evans, G., Housman, D., and Ward, D.C. 1990. High‐resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247:64‐69.
   Lichter, P., Boyle, A.L., Cremer, T., and Ward, D.C. 1991. Analysis of genes and chromosomes by nonisotopic in situ hybridization. Genet. Anal. Techn. Appl. 8:24‐35.
   Manuelidis, L. and Ward, D.C. 1984. Chromosomal and nuclear distribution of the HindIII 1.9 kb human DNA repeat segment. Chromosoma 91:28‐38.
   Mora, J.R., Knoll, J.H.M., Rogan, P.K., Getts, R.C., and Wilson, G.S. 2006. Dendrimer FISH diction of single‐copy intervals in acute promyelocytic leukemia. Mol. Cell Probes 20:114‐120.
   Morrison, L.E., Ramakrishnan, R., Ruffalo, T.M., and Wilber, K.A. 2002. Labeling fluorescence in situ hybridization probes for genomic targets. Methods Mol. Biol. 204:21‐40.
   Morton, C.C., Kirsch, I.R., Taub, R.A., Orkin, S.H., and Brown, J.A. 1984. Localization of the beta‐globin gene by chromosomal in situ hybridization. Am. J. Hum. Genet. 36:576‐585.
   Roda, A., Musiani, M., Pasini, P., Baraldini, M., and Crabtree, J.E. 2000. In situ hybridization and immunohistochemistry with enzyme‐triggered chemiluminescent probes. Methods Enzymol. 305:577‐590.
   Rogan, P.K., Cazcarro, P., and Knoll, J.H.M. 2001. Sequence‐based design of single‐copy genomic DNA probes for fluorescence in situ hybridization. Genome Res. 11:1086‐1094.
   Roth, K.A., Adler, K., and Bobrow, M.N. 1999. 9 enhanced tyramide signal amplification immunohistochemical detection. J. Histochem. Cytochem. 47:1644D‐1645D.
   Shi, S.‐R., Guo, J., Cote, R.J., Young, L.L., Hawes, D., Shi, Y., Thu, S., and Taylor, C.R. 1999. Sensitivity and detection efficiency of a novel two‐step detection system (PowerVision) for immunohistochemistry. Appl. Immunohistochem. Mol. Morphol. 7:201‐208.
   Speel, E.J.M., Schutte, B., Wiegant, J., Ramaekers, F.C., and Hopman, A.H.N. 1992. A novel fluorescence detection method for in situ hybridization, based on the alkaline phosphatase‐fast red reaction. J. Histochem. Cytochem. 40:1299‐1308.
   Trembleau, A., Roche, D., and Calas, A. 1993. Combination of non‐radioactive and radioactive in situ hybridization with immunohistochemistry: A new method allowing the simultaneous detections of two mRNAs and one antigen in the same brain tissue section. J. Histochem. Cytochem. 41:489‐498.
   Van Belkum, A., Linkels, J., Jelsma, T., Van den Berg, F.M., and Quint, W. 1994. Non‐isotopic labeling of DNA by newly developed hapten‐containing platinum compounds. Biotechniques 16:148‐153 [published erratum appears in Biotechniques, 1995, 18:636].
   van de Corput, M.P., Dirks, R.W., van Gijlswijk, R.P., van de Rijke, F.M., and Raap, A.K. 1998. Fluorescence in situ hybridization using horseradish peroxidase–labeled oligodeoxynucleotides and tyramide signal amplification for sensitive DNA and mRNA detection. Histochem. Cell Biol. 110:431‐437.
   Wiegant, J., Tied, T., Nederlof, P.M., van der Ploeg, M., Tanke, H.J., and Raap, A.K. 1991. In situ hybridization with fluoresceinated DNA. Nucl. Acids Res. 19:3237‐3241.
   Zhang, G., Taneja, K., Singer, R.H., and Green, M.R. 1994. Localization of pre‐mRNA splicing in mammalian nuclei. Nature 372:809‐812.
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