Basic Preparative Techniques for Fluorescence In Situ Hybridization

J. Wiegant1, A.K. Raap1

1 Leiden University, Leiden
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.2
DOI:  10.1002/0471142956.cy0802s00
Online Posting Date:  May, 2001
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Abstract

This unit presents protocols for preparing human metaphase chromosome slides from peripheral blood lymphocytes, isolating interphase nuclei from lymphocytes and paraffin‐embedded tissues, and preparing DNA fibers. The protocols are designed so that the resulting preparations are amenable to FISH. The methods correspond to a selection of the specimens that can be analyzed with FISH techniques, and the choice of sample preparation method is highly dependent on the molecular cytogenetics question being addressed.

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Metaphase Chromosome Slides from Peripheral Blood Lymphocytes
  • Basic Protocol 2: Preparation of Slides for Interphase Cytogenetics
  • Basic Protocol 3: Preparation of Slides for DIRVISH
  • Alternate Protocol 1: Preparation of Slides for DIRVISH (Alternative Procedure)
  • Basic Protocol 4: Preparation of DNA Fibers from Fixed Cells
  • Basic Protocol 5: Preparation of DNA Fibers from Agarose Blocks
  • Basic Protocol 6: Preparation of DNA Fibers from Fibroblasts by the HALO Technique
  • Alternate Protocol 2: Preparation of DNA Fibers from Tumor Cells by HALO Technique
  • Basic Protocol 7: Preparation of DNA Fibers by Molecular Combing
  • Support Protocol 1: Preparation of AES‐Coated Slides (General Procedure)
  • Support Protocol 2: Preparation of AES‐Coated Slides for Molecular Combing
  • Support Protocol 3: Preparation of Poly‐L‐Lysine‐Coated Slides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Preparation of Metaphase Chromosome Slides from Peripheral Blood Lymphocytes

  Materials
  • Peripheral blood, collected in 10‐ml heparin‐coated Vacutainer (Becton Dickinson)
  • Chromosome medium with phytohemagglutinin (PAA Laboratories)
  • 0.0025% Colcemid working solution (see recipe)
  • 3 mg/ml thymidine (Sigma) in RPMI 1640 medium, sterile (store ≤3 years at –20°C)
  • Hypotonic buffer (see recipe)
  • Methanol/acetic acid fixative: 3:1 (v/v) absolute methanol/glacial acetic acid (prepare fresh)
  • 100%, 90%, 80%, and 70% acetic acid (optional; if needed)
  • RNase A solution (see recipe)
  • 2× SSC ( appendix 2A), room temperature and 37°C
  • 0.01 M HCl
  • 10% pepsin: dissolve 1 g pepsin (Boehringer Mannheim) in 10 ml H 2O at 37°C; store in aliquots ≤1 year at –20°C
  • 1× PBS (see recipe)
  • Formaldehyde fixative (see recipe)
  • 70%, 90%, and 100% ethanol
  • 250‐ml tissue culture flasks
  • 15‐ml polypropylene centrifuge tubes
  • Tabletop centrifuge (e.g., IEC Clinical or HN‐SII)
  • Microscope slides, cleaned by dipping in 1:1 (v/v) ethanol/diethyl ether and wiping with lint‐free Kimwipes (e.g., Fisher)
  • Phase‐contrast microscope
  • 24 × 50–mm coverslips
  • Rectangular staining dishes (e.g., Fisher)
  • Moist chamber: 1‐liter beaker containing paper towels moistened with 2× SSC ( appendix 2A), covered with aluminum foil

Basic Protocol 2: Preparation of Slides for Interphase Cytogenetics

  Materials
  • Peripheral blood, collected in 10‐ml EDTA Vacutainer (Becton Dickinson) or paraffin‐embedded tissue specimen
  • 1× PBS (see recipe)
  • 0.1 M KCl, 37°C (for blood specimens)
  • 100% methanol (for blood specimens; Baker)
  • Histo‐Clear (for tissue specimens; National Diagnostics)
  • 50%, 70%, 90%, and 100% ethanol
  • 0.1% protease (bacterial type XXIV; Sigma; for tissue specimens) in protease buffer (see recipe)
  • 0.3% (v/v) Nonidet P‐40 (NP‐40) in TE buffer, pH 7.8 ( appendix 2A); use for tissue specimens
  • Methanol/acetic acid fixative: 3:1 (v/v) absolute methanol/glacial acetic acid (prepare fresh); use for tissue specimens (optional)
  • 100% methanol (for tissue specimens)
  • 1:1 (v/v) ethanol/diethyl ether
  • 50% acetic acid
  • 0.1 M Na 2B 4O 7⋅10H 2O, unbuffered (pH 9.3); use for nuclei isolated from tissue specimens
  • RNase A solution (see recipe)
  • 4% formaldehyde (Merck) in 1× PBS (see recipe)
  • 0.01 M HCl
  • 0.1% pepsin (Sigma) in 0.01 M HCl (pH 2.0), 37°C (prepare just before use; for lymphocytes isolated from blood specimens)
  • 1% pepsin (Sigma) in 0.01 M HCl (pH 2.0), 37°C (prepare just before use; for nuclei isolated from tissue specimens)
  • 10‐ml LeucoPrep tubes (for blood specimens; Becton Dickinson)
  • Tabletop centrifuge
  • 15‐ml polypropylene centrifuge tubes
  • 21‐G needle and syringe
  • Microtome with knives (for tissue specimens)
  • 10‐ml glass test tubes (for tissue specimens)
  • 70‐µm nylon‐mesh cell strainer (Falcon)
  • Microscope slides
  • Lint‐free Kimwipes (e.g., Fisher)
  • Hettich‐Universal tabletop cytocentrifuge with #1323 rotor and compatible buckets (#1266, #1271, or #1276) for depositing cells on slides (Hettich‐Zentrifugen)
  • Rectangular staining dishes (e.g., Fisher)
  • 24 × 60–mm coverslips
  • Moist chamber: 1‐liter beaker containing paper towels moistened with 2× SSC ( appendix 2A), covered with aluminum foil

Basic Protocol 3: Preparation of Slides for DIRVISH

  Materials
  • Cells for analysis
  • 1× PBS (see recipe)
  • Lysis buffer (see recipe)
  • Methanol/acetic acid fixative: 3:1 (v/v) absolute methanol/glacial acetic acid (prepare fresh)
  • 70%, 90%, and 100% ethanol
  • RNase A solution (see recipe)
  • Microscope slides: AES‐coated (see protocol 10) or poly‐L‐lysine–coated (see protocol 12)
  • Rectangular staining dishes (e.g., Fisher)
  • Slide box with dessicant

Alternate Protocol 1: Preparation of Slides for DIRVISH (Alternative Procedure)

  • Hair dryer
  • Plant sprayer producing fine mist

Basic Protocol 4: Preparation of DNA Fibers from Fixed Cells

  Materials
  • 70% (v/v) formamide/2× SSC, pH 7 (see appendix 2A for SSC recipe)
  • 70%, 95%, and 100% ethanol
  • 1× PBS (see recipe)
  • NaOH/ethanol: mix 5 vol 0.07 M NaOH with 2 vol 100% ethanol (prepare fresh)
  • 100% methanol
  • Microscope slides: optionally AES‐coated (see protocol 10) or poly‐L‐lysine–coated (see protocol 12)
  • Rectangular staining dishes (e.g., Fisher)
  • Long coverslips
  • Additional reagents and equipment for harvesting and fixing peripheral blood lymphocytes (as for metaphase chromosome slide preparation; see protocol 1)

Basic Protocol 5: Preparation of DNA Fibers from Agarose Blocks

  Materials
  • Cells of interest (e.g., lymphocytes separated by density‐gradient centrifugation or cells from cultured cell lines)
  • 1× PBS (see recipe)
  • 1.9% low gelling/melting temperature agarose (FMC Bioproducts) prepared in 1× PBS (see recipe)
  • Proteinase digestion solution (see recipe)
  • TE buffer ( appendix 2A)
  • Tabletop centrifuge
  • Plexiglas block mold (e.g., Bio‐Rad 10‐well sample‐plug mold)
  • 50°C water bath
  • Poly‐L‐lysine coated microscope slides (see protocol 12)

Basic Protocol 6: Preparation of DNA Fibers from Fibroblasts by the HALO Technique

  Materials
  • Fibroblasts for analysis
  • Dulbecco's modified Eagle medium without phenol red and biotin (Life Technologies)
  • 1× PBS (see recipe), 4°C
  • Solutions A, B, C, D, and E for halo technique (see reciperecipes), 4°C
  • RNase A solution (see recipe)
  • 70%, 90%, and 100% ethanol
  • Microscope slides, cleaned by dipping in 1:1 (v/v) diethyl ether and wiping with lint‐free Kimwipes, then sterilized by heating ≥2 hr at 160°C
  • 50‐ml centrifuge tubes or 100‐ml beakers
  • Glass plate prechilled to 0°C
  • Slide box with dessiccant

Alternate Protocol 2: Preparation of DNA Fibers from Tumor Cells by HALO Technique

  • Tumor tissue
  • 1× PBS (see recipe), 0°C
  • Cryostat
  • 1.5‐ml microcentrifuge tubes
  • AES‐coated microscope slides (see protocol 10)

Basic Protocol 7: Preparation of DNA Fibers by Molecular Combing

  Materials
  • Cosmid, P1, BAC, or YAC DNA prepared for molecular combing (see recipe)
  • 1 × 10–6 M YOYO (Molecular Probes)
  • 22 × 22–mm glass coverslips
  • AES‐coated microscope slides for molecular combing (see protocol 11)

Support Protocol 1: Preparation of AES‐Coated Slides (General Procedure)

  Materials
  • 5% (v/v) aminopropyltriethoxysilane (AES; Sigma) in acetone
  • Microscope slides, cleaned by dipping in 1:1 (v/v) ethanol/diethyl ether and wiping with lint‐free Kimwipes (e.g., Fisher)

Support Protocol 2: Preparation of AES‐Coated Slides for Molecular Combing

  Materials
  • 18 M sulfuric acid
  • 0.1% (v/v) aminopropyltriethoxysilane (AES; Sigma) in ethanol
  • Absolute ethanol
  • Nitrogen source
  • Microscope slides
  • Metal slide holders for dipping slides
  • Rectangular staining dishes
  • 65°C oven
  • Slide box with dessiccant
  • Sealable plastic bags for storing slides under nitrogen atmosphere

Support Protocol 3: Preparation of Poly‐L‐Lysine‐Coated Slides

  Materials
  • 0.2 M HCl
  • Acetone
  • 0.15% (w/v) gelatin/0.3% (w/v) sodium azide (store up to 3 months at 4°C)
  • 0.2% poly‐L‐lysine (Sigma; mol. wt. ∼500,000) in water
  • Microscope slides
  • Rectangular staining dishes
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Figures

Videos

Literature Cited

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   Bensimon, A., Simon, A., Chiffaudel, A., Croquette, V., Heslot, F., and Bensimon, D. 1994. Alignment and sensitive detection of DNA by a moving interface. Science 265:2096‐2098.
   Bentz, M., Cabot, G., Moos, M., Speicher, M.R., Ganser, A., Lichter, P., and Dohner, H. 1994. Detection of chimeric BCR‐ABL genes on bone marrow samples and blood smears in chronic myeloid and acute lymphoblastic leukemia by in situ hybridization. Blood 83:1922‐1928.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7:1513‐1523.
   Bosman, F.T., Van der Ploeg, M., Van Duijn, P., and Schaberg, A. 1975. Chromosome preparations of human blood lymphocytes; evaluation of techniques. Genetica 45:425‐433.
   Carothers, A.D. 1994. Counting, measuring and mapping in FISH‐labelled cells: Sample size considerations and implications for automation. Cytometry 16:298‐304.
   Cremer, T., Landegent, J.E., Brueckner, A., Scholl, H.P., Schardin, M., Hager, H.D., Devilee, P., Pearson, P., and Van der Ploeg, M. 1986. Detection of chromosome aberrations in the human interphase nucleus by visualization of specific target DNAs with radioactive and non‐radioactive in situ hybridization techniques: Diagnosis of trisomy 18 with probe L1.84. Hum. Genet. 74:346‐352.
   Emanuel, B.S. 1993. The use of fluorescence in situ hybridization to identify human chromosomal anomalies. Growth Genet. Hormones 9:6‐12.
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   Kibbelaar, R.E., Kok, F., Dreef, E.J., Kleiverda, J.K., Cornelisse, C.J., Raap, A.K., and Kluin, P.M. 1993. Statistical methods in interphase cytogenetics: An experimental approach. Cytometry 14:716‐724.
   Lewis, J.P., Tanke, H.J., Raap, A.K., Beverstock, G.C., and Kluin‐Nelemans, H.C. 1993. Somatic pairing of centromeres and short arms of chromsome 15 in the hematopoietic and lymphoid system. Hum. Genet. 92:577‐582.
   Lichter, P., Tang, C.C., Call, K., Hermanson, G., Evans, G.A., Housman, D., and Ward, D.C. 1990. High resolution mapping of human chromosome 11 by in situ hybridization with cosmid probes. Science 247:64‐69.
   Oud, P.S., Haag, D.J., Zahniser, D.J., Ramaekers, F.C.S., Huysman, A.C.L.M., Veldhuizen, J.A.M., Verheyen, R.H.M., Verrijp, K., Broers, J.L.V., Herman, C.J., and Vooijs, G.P. 1986. Cytopress: Automated slide preparation of cytologic material from suspensions. Cytometry 7:8‐17.
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   Takahashi, S., Qian, J., Brown, J.A., Alcaraz, A., Bostwick, D.G., Lieber, M.M., and Jenkins, R.B. 1994. Potential markers of prostate cancer aggressiveness detected by fluorescence in situ hybridization in needle biopsies. Cancer Res. 54:3574‐3579.
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   Trask, B., Pinkel, D., and Van Den Engh, G. 1989. The proximity of DNA sequences in interphase nuclei is correlated to genomic distance and permits ordering of cosmids spanning 250 kilobase pairs. Genomics 5:710‐717.
   Vaandrager, J‐W., Schuuring, E., Zwikstra, E., de Boer, C.J., Kleiverda, K.K., van Krieken, J.H.J.M., Kluin‐Nelemans, H.C., van Ommen, G‐J.B., Raap, A.K., and Kluin, P.M. 1996. Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multicolor DNA fiber fluorescence in situ hybridization. Blood 88:1177‐1182.
   Van de Rijke, F.M., Vrolijk, H., Sloos, W.C.R., Tanke, H.J., and Raap, A.K. 1996. Sample preparation and in situ hybridization for automated molecular cytogenetic analysis of white blood cells. Cytometry 24:151‐157.
   Van Driel‐Kulker, A.M.J., Ploem‐Zaaijer, J.J., Van der Zwan‐Van der Zwan, M., and Tanke, H.J. 1980. A preparation technique for exfoliated and aspirated cells allowing different staining procedures. Anal. Quant. Cytol. 2:243‐246.
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Key References
   Arnoldus et al., 1990. See above.
  Along with Tkatchuk et al., , one of the first two reports on translocation detection in interphase cells.
   Carothers, 1994 and Kibbelaar et al., 1993. See above.
  Describes statistical approaches for interphase cytogenetics.
   Cremer et al., 1986. See above.
  Classical article on interphase cytogenetics.
   Emanuel, 1993. See above.
  Elegant overview of FISH.
   Parra and Windle, 1993. See above.
  One of the first reports on fiber‐FISH.
   Rooney, D.E. and Czepulkowski, B.H. (eds.) 1992. Human Cytogenetics. A Practical Approach. Oxford University Press, New York.
  A fully comprehensive manual of established cytogenetic protocols.
   Tkatchuk et al., 1990. See above.
  Along with Arnoldus et al., , one of the first two reports on translocation detection in interphase cells.
   Van de Rijke et al., 1996. See above.
  Describes sample preparation techniques.
   Van Driel‐Kulker et al., 1980. See above.
  Describes bucket centrifugation.
   Verma, R.S. and Babu, A. (eds.) 1995. Human Chromosomes. Principles and Techniques, 2nd ed. McGraw‐Hill, New York.
  Covers the whole field of human (molecular) cytogenetics.
   Vrolijk et al., 1996. See above.
  Describes automated “spot” counter.
   Wiegant et al., 1992. See above.
  One of the first reports on fiber‐FISH.
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