Processing and Staining of Cell and Tissue Material for Interphase Cytogenetics

A.H.M. Hopman1, F.C.S. Ramaekers1

1 University of Maastricht, Maastricht
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 8.5
DOI:  10.1002/0471142956.cy0805s05
Online Posting Date:  May, 2001
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Abstract

This unit describes in detail the protocols for preparation of biological specimens (fresh or fixed) for the purposes of establishing in situ hybridization procedures using probes for specific regions of the genome, allowing targeted detection of numerical and structural chromosome aberrations in the interphase nucleus. The authors present a series of comprehensive protocols from the simplest to the most complex.

Keywords: molecular cytogenetics; in situ hybridization; chromosome aberrations; interphase cytogenetics.

     
 
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Table of Contents

  • Strategic Planning
  • Preparation of Single Cells and Nuclei for ISH
  • Basic Protocol 1: Isolation of Cells by Mechanical Disaggregation of Fresh or Frozen Tissue Samples
  • Basic Protocol 2: Isolation of Nuclei by Enzymatic Treatment of Cell and Tissue Samples
  • Basic Protocol 3: Isolation of Nuclei by Enzymatic Treatment of Formalin‐Fixed and araffin‐Embedded Tissue
  • Preparation of Tissue Sections for ISH
  • Basic Protocol 4: Frozen Tissue Sections
  • Basic Protocol 5: Formalin‐Fixed and Paraffin‐Embedded Tissue Sections
  • Alternate Protocol 1: Formalin‐Fixed and Paraffin‐Embedded Tumor Sections
  • Support Protocol 1: Preparation of Coated Slides
  • Hybridization and Detection
  • Basic Protocol 6: Probe and Target Denaturation and Hybridization
  • Basic Protocol 7: Double‐Target Fluorescence ISH
  • Basic Protocol 8: Double‐Target Bright‐Field ISH
  • Basic Protocol 9: Evaluation and Interpretation of Fluorescence and Bright‐Field ISH Signals
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Cells by Mechanical Disaggregation of Fresh or Frozen Tissue Samples

  Materials
  • 1‐mm3 (or larger) fresh tissue specimen
  • PBS ( appendix 2A)
  • Culture medium (optional)
  • 50 to 400 µg/ml pepsin in 0.01 M HCl (from porcine stomach mucosa, 2500 to 3500 U/mg protein, Sigma; store dilution up to several weeks at 4°C)
  • 1% formaldehyde in PBS (freshly prepared from a 37% formaldehyde stock)
  • 70%, 90%, and 100% ethanol
  • 90‐mm petri dishes
  • 100‐µm nylon filter
  • Poly‐L‐lysine‐ or organosilane‐coated slides (see protocol 7)
  • Shandon Cytospin cytocentrifuge (Life Sciences) or equivalent
  • Coplin jars
  • Additional reagents and equipment for cell counting ( appendix 3A)

Basic Protocol 2: Isolation of Nuclei by Enzymatic Treatment of Cell and Tissue Samples

  Materials
  • Tissue sample: fresh solid tissue block (≤1 mm3; mechanically disaggregate larger blocks, as necessary), tissue fragments (e.g., 30‐ to 50‐µm‐thick frozen sections), or cytological material (e.g., brush material, effusions, chorion villous, or amniotic fluid)
  • 100 µg/ml pepsin in 0.01 M HCl (from porcine stomach mucosa, 2500 to 3500 U/mg protein, Sigma; store dilution up to several weeks at 4°C)
  • 0.01 M HCl
  • 50× TAE ( appendix 2A)
  • 70%, 90%, and 100% ethanol
  • 1% formaldehyde in PBS (freshly prepared from a 37% formaldehyde stock)
  • 21‐G needle
  • Organosilane‐coated slides (see protocol 7)
  • Shandon Cytospin cytocentrifuge (Life Sciences) or equivalent
  • Coplin jars

Basic Protocol 3: Isolation of Nuclei by Enzymatic Treatment of Formalin‐Fixed and araffin‐Embedded Tissue

  Materials
  • 15‐ to 50‐µm‐thick, formalin‐fixed, paraffin‐embedded tissue section
  • 100% xylene
  • 70%, 90%, and 100% ethanol
  • 100% methanol
  • 85% formic acid/0.3% H 2O 2 (prepare fresh)
  • 0.01 M HCl
  • 4 mg/ml pepsin in 0.2 M HCl (store up to several weeks at 4°C)
  • 0.2 M NaOH
  • 21‐G needle
  • Organosilane‐coated slides (see protocol 7)
  • Shandon Cytospin cytocentrifuge (Life Sciences) or equivalent
  • Coplin jars

Basic Protocol 4: Frozen Tissue Sections

  Materials
  • 4‐ to 6‐µm‐thick frozen tissue section
  • 1% formaldehyde in PBS (freshly prepared from a 37% formaldehyde stock)
  • 0.5% Tween 20/ PBS (freshly prepared)
  • 50 to 400 µg/ml pepsin in 0.01 M HCl (from porcine stomach mucosa, 2500 to 3500 U/mg protein, Sigma; store dilution up to several weeks at 4°C)
  • 0.01 M HCl in 70%, 90%, and 100% ethanol (freshly prepared)
  • PBS ( appendix 2A)
  • 70%, 90%, and 100% ethanol
  • Coplin jars
  • Organosilane‐coated slides (see protocol 7)

Basic Protocol 5: Formalin‐Fixed and Paraffin‐Embedded Tissue Sections

  Materials
  • 4‐ to 6‐µm‐thick formalin‐fixed, paraffin‐embedded tissue sections
  • 100% xylene
  • 100% methanol
  • 85% formic acid/0.3% H 2O 2 (prepare fresh)
  • 0.01 M HCl in 70%, 90%, and 100% ethanol
  • 1 M sodium thiocyanate (NaSCN), prewarmed to 80°C (prepare fresh)
  • 4 mg/ml pepsin in 0.02 M HCl (store up to several weeks at 4°C)
  • 0.01 M HCl
  • Organosilane‐coated slides (see protocol 7)
  • Incubator at 56° and 80°C
  • Plastic Coplin jars

Alternate Protocol 1: Formalin‐Fixed and Paraffin‐Embedded Tumor Sections

  • Tumor sections
  • 30% sodium bisulfite (Baker Chemical) in 2× SSC, prewarmed to 45°C (freshly prepared)
  • 2× SSC ( appendix 2A), room temperature and prewarmed to 45°C
  • 20 mg/ml proteinase K in 2× SSC (store aliquots at −20°C)
  • 70%, 90%, and 100% ethanol
  • Incubator at 45°C

Support Protocol 1: Preparation of Coated Slides

  Materials
  • 10% Extran MA01 (EM Science)
  • 1 mg/ml poly‐L‐lysine hydrobromide (MW >150,000; Sigma) in Milli‐Q‐purified H 2O
  • 2% 3‐aminopropyltriethoxysilane (Sigma) in anhydrous acetone
  • Acetone
  • 0.02% NaN 3
  • Glass slides
  • Incubator or water bath at 37°, 60°, and 80°C

Basic Protocol 6: Probe and Target Denaturation and Hybridization

  Materials
  • Hybridization solution (unit 8.3)
  • DNA probe (unit 8.3)
  • Cell or tissue sample (prepared by one of the methods described above; see Basic Protocols protocol 11 to protocol 55)
  • 18 × 18–mm coverslip
  • Additional reagents and equipment for hybridization and for posthybridization washes (unit 8.3)

Basic Protocol 7: Double‐Target Fluorescence ISH

  Materials
  • ISH‐labeled sample with one biotinylated and one digoxigenin‐labeled probe (unit 8.3)
  • 4× SSC ( appendix 2A)/5% nonfat dry milk
  • 1:100 FITC‐conjugated avidin (Vector Labs) in 4× SSC/5% nonfat dry milk
  • 4× SSC ( appendix 2A)/0.05% Tween 20
  • 1:100 biotinylated goat anti‐avidin (Vector Labs) in 4× SSC/5% nonfat dry milk
  • PBS ( appendix 2A)/0.05% Tween 20
  • 1:2000 mouse anti‐digoxin (Sigma) in PBS/0.05% Tween 20/2% normal goat serum
  • 1:100 TRITC‐conjugated rabbit anti–mouse IgG (Dako) in PBS/0.05% Tween 20/2% normal goat serum
  • PBS ( appendix 2A)
  • 70%, 90%, and 100% ethanol
  • Antifading mounting medium (see recipe)
  • Coplin jars
  • 24 × 50–mm coverslips
NOTE: All buffers containing nonfat dry milk, normal goat serum, or antibodies should be prepared fresh.

Basic Protocol 8: Double‐Target Bright‐Field ISH

  Materials
  • ISH‐labeled sample with one biotinylated and one digoxigenin‐labeled probe (unit 8.3)
  • PBS ( appendix 2A)/0.05% Tween 20/2% normal goat serum
  • 1:2000 mouse anti‐digoxin (Sigma) in PBS/0.05% Tween 20/2% normal goat serum
  • PBS ( appendix 2A)/0.05% Tween 20
  • 1:25 alkaline phosphatase (AP)–conjugated goat anti–mouse IgG (Dako) in PBS/0.05% Tween 20/2% normal goat serum
  • PBS ( appendix 2A)
  • New Fuchsin reagent (see recipe)
  • 4× SSC ( appendix 2A)/0.05% Tween 20
  • 1:50 horseradish peroxidase–conjugated avidin (HRP‐avidin; Dako) in 4× SSC ( appendix 2A)/5% nonfat dry milk
  • 3,3′‐diaminobenzidine (DAB) solution (see recipe)
  • 20% hematoxylin (Sigma) in H 2O
  • 50%, 70%, 96%, and 100% ethanol
  • 100% xylene
  • Entellan (organic‐based mounting medium; EM Science)
  • 24 × 50–mm coverslips
  • Coplin jars
NOTE: All buffers containing nonfat dry milk, normal goat serum, or antibodies should be prepared fresh.
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Figures

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Literature Cited

Literature Cited
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   Cremer, T., Landegent, J., Bruckner, 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 nonradioactive in situ hybridization techniques diagnosis of trisomy 18 with probe L1.84. Hum. Genet. 74:346‐352.
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