Fluorescence In Situ Hybridization (FISH)

Jane Bayani1, Jeremy A. Squire1

1 University of Toronto, Ontario, null
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.4
DOI:  10.1002/0471143030.cb2204s23
Online Posting Date:  September, 2004
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Abstract

Fluorescence in situ Hybridization (FISH) involves the preparation of two main components: the DNA probe and the target DNA to which the probe will be hybridized. The DNA probe typically comes from cloned sources such as plasmids, cosmids, PACs, YACs, or BACs; where the insert may contain a specific gene or originate from a specific chromosomal locus. Whole‐chromosome paints may also be used but are usually applicable to metaphase preparations. The purified DNA can then be labeled and detected indirectly using haptens, or labeled directly using fluorochrome or dye‐conjugated nucleotides. Labeling strategies are also variable, employing standard nick translation or PCR labeling methods. The target DNA can take the form of chromosomes spreads or interphase nuclei. The sources of interphase targets may come from cytogenetic preparations or from paraffin‐embedded tissues. Both the labeled DNA probe and DNA target are denatured to a single‐stranded state and permitted to hybridize to each other. Post‐hybridization washes and fluorescently‐labeled antibody incubations follow the 24‐hour hybridization, and the specimen is ready for visualization by fluorescent microscopy. Successful interpretation of FISH experiments is dependent on the quality of the starting materials, hybridization efficiencies, and stringency of post‐hybridization washes and antibody detections.

Keywords: fluorescence in situ hybridization; FISH; slide preparation; probe preparation; stringency

     
 
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Table of Contents

  • Labeling DNA Probes for FISH
  • Basic Protocol 1: Labeling FISH Probes by Nick Translation
  • Alternate Protocol 1: Labeling Probes for FISH by Degenerative Oligonucleotide Primer Polymerase Chain Reaction (DOP‐PCR)
  • Support Protocol 1: Determination of Hapten Incorporation by Dot Blot Analysis
  • Target Slide Preparation
  • Basic Protocol 2: Preparation of Cytogenetic Specimens for FISH
  • Support Protocol 2: Artificial Aging of Cytogenetic Slide Preparations for FISH
  • Alternate Protocol 2: Preparation of Previously G‐Banded Cytogenetic Specimens for FISH
  • Alternate Protocol 3: Preparation of Paraffin‐Embedded Specimens for Hybridization with DNA Probes
  • Alternate Protocol 4: Preparation of Paraffin‐Embedded Specimens for Hybridization with PNA Probes
  • Hybridization
  • Basic Protocol 3: Hybridization of Labeled DNA Probes to Cytogenetic Specimens
  • Alternate Protocol 5: Hybridization of PNA Probes to Cytogenetic Specimens
  • Alternate Protocol 6: Hybridization of Labeled DNA Probes to Sections from Paraffin‐Embedded Material
  • Alternate Protocol 7: Hybridization of PNA Probes to Sections from Paraffin‐Embedded Material
  • Post‐Hybridization Washes and Detection
  • Basic Protocol 4: Post‐Hybridization Washes and Immunofluorescent Detection of Indirectly Labeled DNA Probes
  • Alternate Protocol 8: Post‐Hybridization Washes and Detection of Directly Labeled DNA Probes
  • Alternate Protocol 9: Rapid Wash of Directly Labeled Probes
  • Alternate Protocol 10: Post‐Hybridization Washes for Specimens Hybridized with PNA Probes
  • Interpretation of FISH Findings
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Labeling FISH Probes by Nick Translation

  Materials
  • DNA probe (i.e., cosmid, plasmid, PAC, BAC, or YAC; Table 22.4.1)
  • 10× nick translation buffer (see recipe)
  • DNase I dilution buffer (see recipe)
  • 1 mM dNTP mixture (see recipe)
  • Fluorochrome/dTTP or hapten/dTTP mixture (see reciperecipes)
  • 3 mg/ml DNase I (see recipe)
  • 10 U/µl E. coli DNA polymerase I (Roche)
  • 5 × loading dye (see recipe)
  • 2% (w/v) agarose gel (see recipe)
  • 100‐bp DNA ladder
  • Sonicated salmon sperm DNA standards (i.e., 12.5, 25.0, and 500 ng/µl; see recipe)
  • 1× TBE buffer (see appendix 2A)
  • 300 mM EDTA ( appendix 2A)
  • 10 mg/ml sonicated salmon sperm DNA (Invitrogen)
  • 1 µg/µl human or mouse Cot‐1 DNA (Invitrogen)
  • 3 M sodium acetate ( appendix 2A)
  • 100% ethanol
  • 70% ethanol, cold
  • Hybridization buffer (see recipe)
  • Water bath or PCR machine
  • 0.5‐ml PCR tube (optional)
  • Additional reagents and equipment for agarose gel electrophoresis ( appendix 3A) and determination of hapten incorporation by dot blot analysis (see protocol 3)

Alternate Protocol 1: Labeling Probes for FISH by Degenerative Oligonucleotide Primer Polymerase Chain Reaction (DOP‐PCR)

  • DNA template (Table 22.4.1)
  • 10× PCR buffer: 100 mM Tris⋅Cl, pH 8.3 ( appendix 2A)/500 mM KCl (store up to several months at −20°C)
  • 2 mM dNTP mixture (i.e., dATP, dCTP, dGTP; see recipe)
  • 5 µM each primers 1 and 2 ( appendix 3F)
  • 50 mM MgCl 2
  • 5 to 10 U/µl Taq DNA polymerase
  • Water, sterile
  • Mineral oil, sterile

Support Protocol 1: Determination of Hapten Incorporation by Dot Blot Analysis

  Materials
  • 100 mM Tris·Cl (pH 7.5)/15 mM NaCl ( appendix 2A)
  • Indirectly labeled probe (i.e., with biotin or digoxigenin; Table 22.4.3)
  • Control DNA labeled with biotin or digoxigenin (i.e., labeled DNA probe known to produce good signal strength)
  • 0.5% (w/v) BSA in 100 mM Tris·Cl, pH 7.5 ( appendix 2A)/15 mM NaCl (store up to 6 months at 4°C)
  • AP‐labeled antibody mixture (see recipe)
  • 100 mM Tris·Cl (pH 9.5)/100 mM NaCl/50 mM MgCl 2 ( appendix 2A)
  • NBT/BCIP (see recipe)
  • Charged nylon membrane (∼5 × 5 cm per sample)
  • Filter paper
  • 37°C dry oven
  • Rotating platform

Basic Protocol 2: Preparation of Cytogenetic Specimens for FISH

  Materials
  • Cytogenetic slide preparation (unit 22.2): age naturally at least 2 days at room temperature or artificially (see protocol 5)
  • 10% (w/v) pepsin—100 mg pepsin powder (Sigma) in 1.0 ml H 2O; store in 20‐µl aliquots up to several months at −20°C—and 0.01 M HCl, 37°C or
  • 14 mg/ml proteinase K (Roche Diagnostics) and proteinase K buffer—i.e., 20 mM Tris·Cl, pH 7 ( appendix 2A)/0.2 mM CaCl; store up to several months at room temperature
  • 1× PBS ( appendix 2A)
  • 70% formamide/2× SSC (pH 7.0), 72°C (see unit 18.6 for SSC): prepare fresh
  • 70% ethanol, ice‐cold (for DNA probes)
  • Phase contrast microscope (unit 4.1)
  • Coplin jars
  • Additional reagents and equipment for hybridization (see protocol 9 and protocol 10)
CAUTION: Formamide is a carcinogen and should be handled with care. Discard according to biohazard rules of the institution.NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.4.
Table 2.4.4   Materials   Coplin Jars Needed for Preparation of Cytogenic Specimens for FISH h   Coplin Jars Needed for Preparation of Cytogenic Specimens for FISH

No. Coplin jars Contents Temperature
1 70% ethanol Room temperature
1 70% ethanol j 4°C
1 or 2 j 80% ethanol Room temperature
1 or 2 j 100% ethanol Room temperature
1 70% formamide in 2× SSC j 72°C
1 1× PBS Room temperature
1 Pepsin/0.01 HCl or proteinase K/buffer 37°C or room temperature (respectively)

 hSee protocol 4.
 JFor DNA probes only.

Support Protocol 2: Artificial Aging of Cytogenetic Slide Preparations for FISH

  Materials
  • Banded cytogenetic slide specimen (unit 22.3)
  • Xylene
  • Methanol
  • 70%, 80%, and 100% ethanol
  • 70% formamide in 2× SSC (pH 7.0; unit 18.6), 72°C: prepare fresh
  • 70% ethanol, ice‐cold
  • Coplin jars
NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.7.
Table 2.4.7   Materials   Coplin Jars Needed for Preparation of Previously G‐Banded Cytogenetic Specimens for FISH n   Coplin Jars Needed for Preparation of Previously G‐Banded Cytogenetic Specimens for FISH

No. Coplin jars Contents Temperature
1 70% ethanol 4°C
1 70% ethanol Room temperature
2 80% ethanol Room temperature
2 100% ethanol Room temperature
1 70% formamide/2× SSC, pH 7.0 72°C
1 Methanol Room temperature
1 Xylene Room temperature

 nSee protocol 6.
 CAUTION: Maintain in a fume hood.

Alternate Protocol 2: Preparation of Previously G‐Banded Cytogenetic Specimens for FISH

  Materials
  • 5‐ to 10‐µM paraffin sections on silanized slides
  • Xylene
  • 100% ethanol
  • 0.5% (w/v) pepsin in 0.85% (w/v) NaCl (pH 1.5), 45°C (see recipe)
  • 2× SSC, pH 7.0 (unit )
  • Propidium iodide (PI) or DAPI in antifade (see reciperecipes)
  • 70%, 80% and 100% ethanol
  • Coplin jars
  • 45°C water bath
  • Fluorescence microscope (unit 4.2) with a FITC, and PI or DAPI filter set
NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.8.
Table 2.4.8   Materials   Coplin Jars Needed for Preparation of Paraffin‐Embedded Specimens for Hybridization with DNA Probes   Coplin Jars Needed for Preparation of Paraffin‐Embedded Specimens for Hybridization with DNA Probes

No. Coplin jars Contents Temperature
1 70% ethanol Room temperature
1 80% ethanol Room temperature
3 100% ethanol Room temperature
1 0.5% pepsin in 0.85% NaCl 45°C
4 2× SSC, pH 7.0 Room temperature
2 Xylene Room temperature

 See protocol 7.
 CAUTION: Maintain in a fume hood.

Alternate Protocol 3: Preparation of Paraffin‐Embedded Specimens for Hybridization with DNA Probes

  Materials
  • 5‐ to 10‐µM paraffin sections on silanized slides
  • Xylene
  • 100% ethanol
  • 100 µg/ml RNase I (see recipe)
  • 2× SSC (unit 18.6)
  • 1 M sodium thiocyanate, 80°C
  • 5 mg/ml pepsin in 0.85% (w/v) NaCl, 45°C (see recipe)
  • 0.1 M triethanolamine, pH 8.0
  • Acetic anhydride (Sigma)
  • 1× PBS ( appendix 2A)
  • 70%, 80%, and 100% ethanol
  • 45°C hot plate or slide warmer
  • Coplin jars
  • 37°C oven
NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.10.
Table 2.4.0   Materials   Coplin Jars Needed for Preparation of Paraffin‐Embedded Specimens for Hybridization with PNA Probes   Coplin Jars Needed for Preparation of Paraffin‐Embedded Specimens for Hybridization with PNA Probes

No. Coplin jars Contents Temperature
1 70% ethanol Room temperature
1 80% ethanol Room temperature
4 100% ethanol Room temperature
2 1× PBS Room temperature
1 5 mg/ml pepsin in 0.85% NaCl 45°C
1 1 M sodium thiocyanate 80°C
5 2× SSC Room temperature
1 Triethanolamine, pH 8.0 Room temperature
2 Water Room temperature
3 Xylene Room temperature

 See protocol 8.
 CAUTION: Maintain in a fume hood.

Alternate Protocol 4: Preparation of Paraffin‐Embedded Specimens for Hybridization with PNA Probes

  Materials
  • DNA probe (see protocol 1 and protocol 2)
  • Pretreated and denatured cytogenetic slide specimen (see protocol 4 and protocol 5)
  • Rubber cement
  • 75°C water bath or PCR machine
  • 37°C dry oven
  • Glass coverslips
  • Hybridization box, slightly dampened (e.g., black plastic video tape box lined with slightly moistened gauze or paper towel)
  • Hybridization container (i.e., any plastic container with lid or a black video cassette box)

Basic Protocol 3: Hybridization of Labeled DNA Probes to Cytogenetic Specimens

  • Labeled PNA probe (Applied Biosystems)
  • Pretreated cytogenetic specimen, not denatured ( protocol 4)
  • 80°C oven, hot plate, or HYBrite (Vysis)

Alternate Protocol 5: Hybridization of PNA Probes to Cytogenetic Specimens

  • Deparaffinized and enzyme‐digested specimen (see protocol 7)
  • 90°C oven, hot plate, or HYBrite (Vysis)

Alternate Protocol 6: Hybridization of Labeled DNA Probes to Sections from Paraffin‐Embedded Material

  • PNA probes (Applied Biosystems)
  • Deparaffinized and enzyme‐digested specimen (see protocol 8)
  • 80°C oven, hot plate or HYBrite (Vysis)

Alternate Protocol 7: Hybridization of PNA Probes to Sections from Paraffin‐Embedded Material

  Materials
  • Hybridized slides in a hybridization box (see protocol 9 and protocol 11)
  • 50% formamide in 2× SSC, 45°C (prepare fresh)
  • 1× SCC, 45°C (unit 18.6)
  • Blocking solution: 1% BSA (w/v)/0.1% (v/v) Tween 20 in 4× SSC (store indefinitely at −20°C or up to several months at 4°C)
  • Primary, secondary, and tertiary antibodies (see recipe for antibodies)
  • 0.1% (v/v) Tween 20 in 4× SSC, 45°C
  • DAPI in antifade (see recipe)
  • Clear nail polish
  • Coplin jars
  • 22 × 50–mm glass coverslips
  • 37°C oven
  • Fluorescent microscope and appropriate filter sets (unit 4.2)
CAUTION: Formamide is a carcinogen and should be handled with care. Discard according to biohazard rules of the institution.NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.13.
Table 2.4.3   Materials   Coplin Jars Needed for Post‐Hybridization Washes and Immunofluorescent Detection of Indirectly Labeled DNA Probes   Coplin Jars Needed for Post‐Hybridization Washes and Immunofluorescent Detection of Indirectly Labeled DNA Probes

No. Coplin jars Contents Temperature
3 50% formamide in 2× SSC 45°C
3 1× SSC 45°C
9 0.1% Tween 20 in 4× SSC 45°C

 See protocol 13.

Basic Protocol 4: Post‐Hybridization Washes and Immunofluorescent Detection of Indirectly Labeled DNA Probes

  • 2× SSC (unit 18.6)
  • 0.3% (v/v) NP‐40 in 2× SSC, room temperature and 72°C
NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.17.
Table 2.4.7   Additional Materials (also see protocol 13)   Additional Materials   Coplin Jars Needed for Rapid Wash of Directly Labeled Probes   Coplin Jars Needed for Rapid Wash of Directly Labeled Probes

No. Coplin jars Contents Temperature
1 0.3% (v/v) NP‐40 in 2× SSC 72°C
1 0.3% (v/v) NP‐40 in 2× SSC Room temperature
1 2× SSC Room temperature

 See protocol 15.

Alternate Protocol 8: Post‐Hybridization Washes and Detection of Directly Labeled DNA Probes

  Materials
  • Slides hybridized with PNA probes (see protocol 10 or protocol 127)
  • 70% (v/v) formamide/10 mM Tris·Cl (pH 7.0 to 7.5)/0.1% (w/v) BSA (see recipe)
  • 0.1 M Tris·Cl (pH 7.0 to 7.5)/0.15 M NaCl/0.08% (v/v) Tween 20 (store up to several weeks at room temperature)
  • 70%, 90%, and 100% ethanol
  • DAPI in antifade solution (see recipe)
  • Fluorescent microscope and appropriate filters
NOTE: A list of all Coplin jars used in this protocol is given in Table 22.4.18.
Table 2.4.8   Materials   Coplin Jars Needed for Post‐Hybridization Washes for Specimens Hybridized with PNA Probes   Coplin Jars Needed for Post‐Hybridization Washes for Specimens Hybridized with PNA Probes

No. Coplin jars Contents Temperature
2 70% formamide/10 mM Tris·Cl (pH 7.0–7.5)/0.1% BSA Room temperature
3 0.1 M Tris·Cl (pH 7.0–7.5)/0.15 M NaCl/0.08% Tween 20 Room temperature
1 70% ethanol Room temperature
1 90% ethanol Room temperature
1 100% ethanol Room temperature

 See protocol 16.
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Figures

  •   FigureFigure 22.4.1 Nick translated DNA fragments electrophoresed on a 2% agarose gel. A volume of 10.0 µl labeled product was loaded. The fragment sizes range from 200 to 500 bp as determined from the molecular marker. The estimated concentration of the probe is 200 ng in 10 µl, yielding ∼20 ng/µl.
  •   FigureFigure 22.4.2 Dot‐blot analysis of biotin incorporation in probes shown in Figure . Compared to control labeled DNA, the labeled probes appear to have incorporated biotin well, with probe 4 showing the greatest incorporation compared to probe 1. The concentration of each probe was estimated at 20 ng/µl; however, it is evident that the labeling efficiency was greater in some probes over others.
  •   FigureFigure 22.4.3 Digestion of paraffin‐embedded specimen for FISH. (A) Under‐digestion is indicated by weak uptake of the DAPI stain. (B) Increased digestion permits better access to the DNA and increase uptake of DAPI.
  •   FigureFigure 22.4.4 Composite figure illustrating FISH hybridization. (A) Assessment of a DNA probe. (i) Cross‐hybridization of probe (arrowhead) to chromosome 1. True signals are located on chromosome 22. (ii) Changes in stringency washes and post‐hybridization removes cross‐hybridization. (iii) High background associated with a insufficient repetitive sequence (Cot‐1) suppression in this mouse metaphase spread. Weak signals (arrows) indicate poor labeling efficiency. (B) Interphase FISH analysis. (i) A cytogenetic specimen from a short‐term ovarian primary culture hybridized with PNA probes for centromeres 7 (green) and 8 (red). When scoring interphase nuclei, it is especially important to focus through the cells since signals may be present at different z planes. (ii) Schematic indicating the nuclei that are acceptable for scoring: A, cell acceptable for scoring; B, cell is likely acceptable for scoring, but requires careful attention; ?, cell has qualities that make it questionable for scoring; X, cell is not acceptable for scoring. (iii) Interphase analysis using PNA probes specific for centromeres 7(green) and 8 (red) on a paraffin section from a prostate carcinoma. Arrowheads indicate cells containing changes in ploidy. (C) Analysis of translocation probes. (i) and (ii) Results of consecutive hybridization of a G‐banded metaphase spread with the Vysis BCR/ABL translocation probe. Two fusion signals (yellow) are produced from the hybridization of BCR/ABL fusion on the Philadelphia chromosomes (Ph) on chromosome 22 and the reciprocal ABL/BCR on the derivative chromosome 9. Separate green and red signals from the normal chromosomes 9 and 22 are also seen. (iii) Interphase pattern from this specimen. (D) Example of a chromosomal inversion on chromosome 11. An inversion was identified involving the terminal portion of chromosome 11 by gross cytogenetic analysis. Clones 200 kb apart and in the 11p15.5 region, containing the IGF2 gene (green) and H19 (red) were hybridized to the patient specimen. The normal chromosome 11 shows the red and green signal hybridizing on top of each other at 11p15.5. The inverted 11 shows the clear spit of signal along the abnormal chromosome 11 indicating the breakpoint lies within the 200 kb between IGF2 and H19. (E) Gene amplification of MYCN in neuroblastoma specimens. (i) Double minute chromosomes (dmns) containing hundreds of copies of the MYCN gene. This is in contrast to amplification of MYCN in (ii) as a large block of signal called a homogeneously staining region (HSR). Interphase nuclei nearby show the typical hybridization pattern for an HSR. (F) Use of subtelomeric and pan‐centromeric PNA probes. (i) Hybridization of a prostate cell line with PNA subtelomeric probes. (ii) Hybridization of another prostate cell line with subtelomeric and pan‐centromeric PNA probes. Loss of telomeric sequences are indicated by the arrow while the presence of multicentric chromosomes are indicated by arrow heads.
  •   FigureFigure 22.4.5 Schematic representation of scoring criteria. Shown in the illustration are typical hybridization signal configurations. These images pertain to a single probe, such as a centromere‐ or locus‐specific probe. Signals must be more than one signal width apart to be considered one signal. Signals joined by a string of hybridization are also considered as one signal.

Videos

Literature Cited

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   Beatty, B., Mai, S., and Squire, J.A. 2002. FISH: A Practical Approach. Oxford University Press, New York.
   Boyle, A.L., Ballard, S.G., and Ward, D.C. 1990. Differential distribution of long and short interspersed element sequences in the mouse genome: Chromosome karyotyping by fluorescence in situ hybridization. Proc. Natl. Acad. Sci. U.S.A. 87:7757‐7761.
   Ghadimi, B.M., Heselmeyer‐Haddad, K., Auer, G., and Ried, T. 1999. Interphase cytogenetics: At the interface of genetics and morphology. Anal. Cell Pathol. 19:3‐6.
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   Qian, J., Bostwick, D.G., Takahashi, S., Borell, T.J., Brown, J.A., Lieber, M.M., and Jenkins, R.B. 1996. Comparison of fluorescence in situ hybridization analysis of isolated nuclei and routine histological sections from paraffin‐embedded prostatic adenocarcinoma specimens. Am. J. Pathol. 149:1193‐1199.
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Internet Resources
   http://www.appliedbiosystems.com
  The Applied Biosystems website. This company supplies PNA probes and custom orders PNA probes.
   http://www.cytocell.co.uk
  The Cytocell website. This is a commercial supplier of DNA FISH probes
   http://www.biochem.roche.com
  The Roche Molecular Biochemicals website. This company is a supplier of antibody and labeling reagents.
   http://www.vysis.com
  The Vysis website. This company is a supplier of commercial DNA FISH probes.
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