Tyramide Signal Amplification (TSA) Systems for the Enhancement of ISH Signals in Cytogenetics

Mark N. Bobrow1, Philip T. Moen1

1 NEN Life Science Products, Boston, Massachusetts
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.9
DOI:  10.1002/0471142956.cy0809s11
Online Posting Date:  May, 2001
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Abstract

This unit provides a series of techniques that dramatically improve the sensitivity of direct and indirect in situ detection. TSA is a peroxidase‐based signal amplification system which is compatible with all in situ hybridization as well as immunocytochemical detection systems. The assay is performed on a glass slide and combines the use of fluorescent probes in either direct or indirect format using either an enzyme‐labeled streptavidin or antibody. Protocols are provided for brightfield and fluorescent detection of single DNA targets, DNA or RNA in cultured cells, multitarget detection for DNA/RNA and DNA and RNA. In addition a protocol is included for the TSA plus system, a DNP‐based detection system using horseradish peroxidase and alkaline phosphatase.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Chromogenic Detection Using TSA‐Indirect
  • Alternate Protocol 1: Chromogenic Detection Using the TSA Plus System with Horseradish Peroxidase
  • Alternate Protocol 2: Chromogenic Detection Using the TSA Plus System with Alkaline Phosphatase
  • Basic Protocol 2: Fluorescence Detection Using TSA‐Indirect
  • Basic Protocol 3: Fluorescence Detection Using TSA‐Direct
  • Basic Protocol 4: Fluorescence Detection of DNA or RNA in Cultured Cells
  • Alternate Protocol 3: Fluorescence Detection of Multiple DNA Targets in Cultured Cells
  • Alternate Protocol 4: Fluorescence Detection of Multiple RNA Targets in Cultured Cells
  • Alternate Protocol 5: Fluorescence Detection of DNA and RNA Targets in Cultured Cells
  • Support Protocol 1: Preparation of Cultured Cells for Hybridization
  • Support Protocol 2: Fixation and Permeabilization of Cultured Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Chromogenic Detection Using TSA‐Indirect

  Materials
  • HRP‐labeled ISH preparation: nucleic acid target hybridized with hapten‐labeled probe (unit 8.2 & ), followed by the immunocytochemical incorporation of HRP (unit 8.4)
  • TSA‐Indirect (ISH) Kit (NEN Life Sciences), including:
  •  Stock streptavidin‐HRP reagent
  •  Blocking reagent (used for TNB buffer)
  •  Amplification diluent
  •  Tyramide stock reagent (see recipe): biotinyl‐tyramide
  • TNT buffer (see recipe)
  • TNB buffer (see recipe)
  • 2.5% (w/v) 3,3′‐diaminobenzidine (DAB) solution (NEN Life Sciences)
  • 30% (v/v) H 2O 2 (Sigma)
  • DAB buffer (see recipe)
  • Additional reagents and equipment for counterstaining, drying, and mounting the slides (units 8.3 & 8.4)

Alternate Protocol 1: Chromogenic Detection Using the TSA Plus System with Horseradish Peroxidase

  • TSA Plus (HRP) System (NEN Life Sciences), including:
  •  Dinitrophenyl (DNP) stock amplification reagent (see product manual)
  •  1× Plus amplification diluent
  •  Blocking reagent (used for TNB buffer)
  •  Horseradish peroxidase–labeled anti‐DNP antibody (anti‐DNP‐HRP)

Alternate Protocol 2: Chromogenic Detection Using the TSA Plus System with Alkaline Phosphatase

  • TSA Plus (AP) System (NEN Life Sciences), including:
  •  Dinitrophenyl (DNP) stock amplification reagent (see product manual)
  •  1× Plus amplification diluent
  •  Blocking reagent (used for TNB buffer)
  •  Alkaline phosphatase–labeled anti‐DNP antibody (anti‐DNP‐AP)
  • AP substrate solution (unit 8.4)

Basic Protocol 2: Fluorescence Detection Using TSA‐Indirect

  Materials
  • HRP‐labeled ISH preparation: nucleic acid target hybridized with hapten‐labeled probe (units 8.2 & 8.3), followed by the immunocytochemical incorporation of HRP (unit 8.4)
  • TSA‐Indirect (ISH) Kit (NEN Life Sciences), including:
  •  Blocking reagent (used for TNB buffer)
  •  Amplification diluent
  •  Tyramide stock reagent (see recipe): biotinyl‐tyramide
  • Fluorescent streptavidin conjugate: fluorescein, Texas Red, or coumarin (NEN Life Sciences), or any fluorescent streptavidin conjugates available from Molecular Probes (e.g., Alexa fluors) and other vendors (e.g., cyanine dyes)
  • TNT buffer (see recipe)
  • TNB buffer (see recipe)
  • Additional reagents and equipment for counterstaining, drying, and mounting the slides (units 8.3 & 8.4)

Basic Protocol 3: Fluorescence Detection Using TSA‐Direct

  Materials
  • HRP‐labeled ISH preparation: nucleic acid target hybridized with hapten‐labeled probe (units 8.2 & 8.3), followed by the immunocytochemical incorporation of HRP (unit 8.4)
  • TSA‐Direct (ISH) Kit (NEN Life Sciences), including:
  • Amplification diluent
  • Tyramide stock reagent (see recipe): fluorochrome‐tyramide, e.g., fluorescein (green), tetramethylrhodamine (red), coumarin (blue), cyanine 3 (red), or cyanine 5 (far red)
  • TNT buffer (see recipe)
  • Additional reagents and equipment for counterstaining, drying, and mounting the slides (unit 8.3 & )

Basic Protocol 4: Fluorescence Detection of DNA or RNA in Cultured Cells

  Materials
  • 10 µg/ml labeled probe preparation
  • 1 mg/ml Cot‐1 DNA (Life Technologies)
  • 10 mg/ml sheared salmon sperm DNA (Sigma)
  • 10 mg/ml yeast tRNA (Sigma)
  • Formamide (Sigma)
  • RNA hybridization buffer (see recipe)
  • Vanadyl ribonucleoside complex (VRC; New England Biolabs)
  • DNA hybridization buffer (see recipe)
  • Cells prepared for hybridization (see protocol 10)
  • 4× SSC ( appendix 2A), filtered through a 0.2‐µm filter unit
  • SSC/50% (v/v) formamide (Sigma), pH 7.5, freshly prepared and prewarmed to 37°C
  • SSC/0.1% (v/v) Triton X‐100 (Sigma)
  • Speedvac evaporator
  • 95°C heating block or water bath
  • Forceps
  • Humidified chamber
  • Coplin jar
  • Additional reagents and equipment for incorporating HRP into ISH signals (units 8.3 & 8.4), for preparing working tyramide solution (see 8.9), and for dehydrating, mounting, and counterstaining slides (units 8.3 & 8.4)

Alternate Protocol 3: Fluorescence Detection of Multiple DNA Targets in Cultured Cells

  • Two differentially labeled, 10 µg/ml probes
  • Two appropriate horseradish peroxidase (HRP) conjugates (e.g., streptavidin‐HRP and anti‐digoxigenin‐HRP)
  • Two appropriate tyramide solutions (e.g., cyanine 3 tyramide and fluorescein tyramide; see protocol 5)
  • Sodium acetate/sodium azide buffer (see recipe)
  • H 2O 2 (Sigma)

Alternate Protocol 4: Fluorescence Detection of Multiple RNA Targets in Cultured Cells

  • Two differentially labeled, 10 µg/ml hybridization probes

Alternate Protocol 5: Fluorescence Detection of DNA and RNA Targets in Cultured Cells

  Materials
  • Fixed, permeabilized cells (see protocol 11)
  • Absolute and 70% (v/v) ethanol
  • 2× SSC ( appendix 2A), filtered through a 0.2‐µm filter unit
  • SSC/70% (v/v) formamide (deionized; Sigma), freshly prepared, pH 7.2
  • 0.07 N NaOH/70% ethanol
  • RNase cocktail (Ambion)
  • 50 mM Tris⋅Cl, pH 8.0 ( appendix 2A)/5 mM MgCl 2, filtered through a 0.2‐µm filter unit

Support Protocol 1: Preparation of Cultured Cells for Hybridization

  Materials
  • Cells grown on or deposited onto a microscope slide or glass coverslip
  • HBSS ( appendix 2A)
  • Permeabilizing solution (select one):
  •  CSK/Triton X‐100 solution (see recipe)
  •  CSK/Triton X‐100/VRC solution (see recipe)
  •  DPBS/Triton X‐100/VRC solution (see recipe)
  • 4% (w/v) paraformaldehyde solution (see recipe)
  • DPBS (see recipe)
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Figures

Videos

Literature Cited

   Bobrow, M.N. and Litt, G.J., March 1993. Method for the detection or quantitation of an analyte using an analyte dependent enzyme activation system. U.S. patent 5,196,306.
   Bobrow, M.N. and Litt, G.J., December 1996. Method for the detection or quantitation of an analyte using an analyte dependent enzyme activation system. U.S. patent 5,583,001.
   Bobrow, M.N. and Litt, G.J., March 1998. Catalyzed reporter deposition. U.S. patent 5,731,158.
   Bobrow, M.N., Harris, T.D., Shaughnessy, K.J., and Litt, G.J. 1989. Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays . J. Immunol. Methods 125:279‐285.
   Bobrow, M.N., Shaughnessy, K.J., and Litt, G.J. 1991. Catalyzed reporter deposition, a novel method of signal amplification. Application to membrane immunoassays. J. Immunol. Methods 137:103‐112.
   Bobrow, M.N., Litt, G.J., Shaughnessy, K.J., Mayer, P.C., and Conlon, J. 1992. The use of catalyzed reporter deposition as a means of signal amplification in a variety of formats. J. Immunol. Methods 150:145‐149.
   Mayer, G. and Bendayan, M. 1997. Biotinyl‐tyramide: A novel approach for electron microscopic immunocytochemistry. J. Histochem. Cytochem. 45:1449‐1454.
   Mayer, P. and Nayak, Y. 1999. Recent developments in signal amplification. Am. Biotechnol. Lab. 17:18‐20.
   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.
   van Gijlswijk, R.P.M., Wiegant, J., Raap, A.K., and Tanke, H.J. 1996a. Improved localization of fluorescent tyramides for fluorescence in situ hybridization using dextran sulfate and polyvinyl alcohol. J. Histochem. Cytochem. 44:389‐392.
   van Gijlswijk, R.P.M., Wiegant, J., Vervenne, R., Lasan, R., Tanke, H.J., and Raap, A.K. 1996b. Horseradish peroxidase‐labeled oligonucleotides and fluorescent tyramides for rapid detection of chromosome‐specific repeat sequences. Cytogenet. Cell Genet. 75:258‐262.
   van Gijlswijk, R.P.M., Zijlmans, H.J.M.A.A., Wiegant, J., Bobrow, M.N., Erickson, T.J., Adler, K.E., Tanke, H.J., and Raap, A.K. 1997. Fluorochrome‐labelled tyramides: Use in immunocytochemistry and fluorescence in situ hybridization. J. Histochem. Cytochem. 45:375‐382.
   Yang, H., Wanner, I.B., Roper, S.D., and Chaudhari, N. 1999. An optimized method for in situ hybridization with signal amplification that allows the detection of rare mRNAs. J. Histochem. Cytochem. 47:431‐445.
   Zehbe, I., Hacker, G.H., Su, H., Hauser‐Kronberger, C., Hainfeld, J.H., and Tubbs, R. 1997. Sensitive in situ hybridization with catalyzed reporter deposition, Streptavidin‐Nanogold, and silver acetate autometallography. Am. J. Path. 150:1553‐61.
Key References
   Mayer and Bendayan, 1997. See above.
  Electron microscopy.
   Villnave‐Johnson, C., Singer, R.H., and Lawrence, J.B. 1991. Fluorescent detection of nuclear RNA and DNA: Implications for genome organization. Methods Cell Biol. 35:73‐99.
  In situ hybridization to DNA and RNA.
   Xing, Y., Johnson, C.V., Moen, P.T. Jr., McNeil, J.A., and Lawrence, J.B. 1995. Nonrandom gene organization: Structural arrangements of specific pre‐mRNA transcription and splicing with SC‐35 domains. J. Cell Biol. 131:1635‐1647.
  Simultaneous detection of DNA and RNA in situ.
   Yang, et al., 1999. See above.
  Optimization of hybridization and detection conditions.
   Zehbe et al., 1997. See above.
  Silver‐enhanced gold detection.
Internet Resources
   http://www.nen.com
  The most current information for reagents, protocols, and applications.
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