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, null, 2 Deutsches Krebsforschungszentrum, Heidelberg, null, 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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Abstract

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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Materials

Basic Protocol 1: Fluorescence in Situ Hybridization

  Materials
  • 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

  Materials
  • 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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Figures

  •   FigureFigure 14.7.1 Detection of hybridized probe by (A) fluorescence in situ hybridization (FISH) and (B) enzymatic detection. Abbreviations: e, enzyme (e.g., alkaline phosphatase or horseradish peroxidase); f, fluorochrome (e.g., fluorescein, rhodamine, or Texas red); p, colored precipitate product; pr, probe labeled with reporter molecule; r, reporter molecule (e.g., biotin, digoxigenin); r‐b, reporter‐binding molecule (e.g., avidin, streptavidin, or digoxigenin antibody); s, soluble substrate; tr, transcript. Arrow indicates reaction catalyzed by enzyme.
  •   FigureFigure 14.7.2 Enzyme‐mediated detection of reporter molecules. (A) For direct detection, enzyme is conjugated to reporter‐binding molecule. (B) For a two‐step procedure, reporter‐binding molecule is applied first, followed by an incubation with reporter‐conjugated enzyme. (C) For signal amplification, incubation with reporter‐binding molecule is followed by incubation with an antibody to reporter‐binding molecule. The antibody may be conjugated with enzyme or reporter molecule. In the latter case, an incubation with enzyme‐conjugated reporter‐binding molecule follows. The final step is addition of substrate (not shown). Fluorochrome can be substituted for enzyme molecule. Abbreviations: ab, antibody to reporter‐binding molecule, conjugated with enzyme or reporter molecule; e, enzyme (e.g., alkaline phosphatase or horseradish peroxidase); pr, probe labeled with reporter molecule; r, reporter molecule (e.g., biotin or digoxigenin); r‐b, reporter‐binding molecule (e.g., avidin, streptavidin, or anti‐digoxigenin); tr, transcript.

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Literature Cited

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