Preparation of Cells from Formalin‐Fixed, Paraffin‐Embedded Tissue for Use in Fluorescence In Situ Hybridization (FISH) Experiments

Stanislawa Weremowicz1

1 CAMD‐Cytogenetics, Department of Pathology, Brigham and Women's Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.8
DOI:  10.1002/0471142905.hg0808s84
Online Posting Date:  January, 2015
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Abstract

Numerical and structural chromosome abnormalities can be accurately detected in cells from archived tissues using fluorescence in situ hybridization (FISH). This unit describes two common approaches to performing FISH in formalin‐fixed, paraffin‐embedded tissue. The first approach utilizes 4 to 6 μm tissue sections in cases for which preserving tissue morphology is necessary, and the second involves extraction of intact nuclei from 50‐μm tissue sections. To interpret FISH results using 4 to 6 μm sections, an adequate number of nuclei must be evaluated to perform statistical analysis. Evaluation of 30 to 50 nuclei from the single‐cell suspension generally gives an interpretable result. © 2015 by John Wiley & Sons, Inc.

Keywords: interphase cytogenetics; FISH; archived tissue

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Paraffin Sections for FISH
  • Basic Protocol 2: Preparation of Single‐Cell Suspension from Formalin‐Fixed, Paraffin‐Embedded Tissue for FISH
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Paraffin Sections for FISH

  Materials
  • Paraffin‐embedded tissue of interest
  • Silanized glass (precleaned), or positively charged, microscope slides (see recipe)
  • Xylene or Hemo‐De (Scientific Safety Solvents)
  • Ethanol
  • Sodium bisulfite (Sigma‐Aldrich)
  • 2× sodium chloride/sodium citrate (SSC), pH 7.0 (see appendix 2D for 20× SSC)
  • 25 mg/ml proteinase K (Sigma‐Aldrich; store at –20°C) or pepsin (Paraffin Pretreatment Kit 1, Abbott Molecular; store at –20°C)
  • 0.6 μg/ml propidium iodide/Antifade (MP Biomedicals)
  • DNA probes (Abbott Molecular, MP Biomedicals, Cytocell Ltd.)
  • Hybridization buffer (MP Biomedicals; LSI or CEP, Abbott Molecular)
  • Rubber cement
  • 0.3% (v/v) Tween 20 in 0.4× SSC (pH 7.0)
  • 0.1% (v/v) Tween 20 in 2× SSC (pH 7.0)
  • Phosphate‐buffered detergent (PBD; MP Biomedicals), room temperature
  • 125 mg/ml 4′,6‐diamidino‐2‐phenylindole (DAPI) II/Antifade solution (Abbott Molecular)
  • Filter set:
    • Green/Red dual band (e.g., cat. no. 59010, Chroma Technology Corp.; see also unit 4.4; McNamara et al., )
    • Aqua/Green/Red triple band (e.g., cat. no. 69008, Chroma Technology Corp.; see also unit 4.4; McNamara et al., )
  • Microtome
  • 65°C and 95°C ovens (or a slide warmer; e.g., HYBrite, Abbott Molecular, Leica Biosystems)
  • 50‐ml glass Coplin jars
  • Epifluorescence microscope equipped with 100‐W mercury lamp (unit 4.4; McNamara et al., )
  • 20× objective and 40× or 100× oil immersion fluorescence objectives
  • 22 × 22‐mm glass coverslips
  • Humidified hybridization chamber (unit 4.3; Knoll and Lichter, )
  • 24 × 50‐mm glass no.1 coverslips
  • Additional reagents and equipment for paraffin embedding and preparation of tissue sections (Zeller, ), FISH (unit 4.3; Knoll and Lichter, ), and fluorescence microscopy (unit 4.4; McNamara et al., )

Basic Protocol 2: Preparation of Single‐Cell Suspension from Formalin‐Fixed, Paraffin‐Embedded Tissue for FISH

  Materials
  • Paraffin‐embedded tissue of interest
  • Silanized glass (precleaned), or positively charged, microscope slides (see recipe)
  • Xylene
  • Ethanol
  • Hanks’ balanced salt solution (HBSS; appendix 2D)
  • 1 mg/ml collagenase XI solution (Sigma‐Aldrich)
  • 0.05% trypsin/EDTA (Invitrogen)
  • Sterile H 2O
  • Microtome
  • 15‐ml glass conical centrifuge tubes
  • Centrifuge (e.g., Marathon 3000, Fisher Scientific)
  • Vortex
  • 50°C oven (or slide warmer; e.g., HYBrite, Abbott Molecular, Leica Biosystems)
  • Additional reagents and equipment for probe hybridization (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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