Visualization of Telomere Integrity and Function In Vitro and In Vivo Using Immunofluorescence Techniques

Anthony J. Cesare1, Christopher M. Heaphy2, Roderick J. O'Sullivan3

1 Children's Medical Research Institute, The University of Sydney, Westmead, 2 Johns Hopkins University School of Medicine, Baltimore, Maryland, 3 University of Pittsburgh Cancer Institute, Hillman Cancer Center, Pittsburgh, Pennsylvania
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.40
DOI:  10.1002/0471142956.cy1240s73
Online Posting Date:  July, 2015
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Abstract

In cancer cells, telomere length maintenance occurs largely via the direct synthesis of TTAGGG repeats at chromosome ends by telomerase, or less frequently by the recombination‐dependent alternative lengthening of telomeres (ALT) pathway. The latter is characterized by the atypical clustering of telomeres within promyelocytic leukemia (PML) nuclear bodies, which harbor proteins that are linked with DNA repair and recombination activity. For this reason, it is speculated that these associated PML bodies represent the sites of the recombination that maintains telomere length. The protocols described here can be employed for the routine investigation of the structural integrity of telomeres and the association of proteins at telomeres in normal cells, challenged cells, and archived formalin‐fixed paraffin‐embedded clinical tissue specimens that may have activated the ALT pathway. © 2015 by John Wiley & Sons, Inc.

Keywords: telomeres; metaphase; DNA damage; ALT pathway

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Combined Immunofluorescence and Telomere Fluorescence In Situ Hybridization (FISH) on Fixed Adherent Cells
  • Support Protocol 1: Combined Immunofluorescence and Telomere Fluorescent In Situ Hybridization (FISH) on Cytocentrifuged Chromosome Spreads (Metaphase‐TIF Assay)
  • Basic Protocol 2: In Vivo Detection of Telomeres in Archived Clinical Specimens
  • Support Protocol 2: In Vivo Detection of APBS in Archived Clinical Specimens
  • Basic Protocol 3: Detection of Telomeric DNA Sequence on Mitotic Chromosome Spreads with Fluorescent In Situ Hybridization (FISH)
  • Support Protocol 3: Detection of Telomere Sister Chromatid Exchange (T‐SCE) on Mitotic Chromosome Spreads by Chromosome Orientation FISH (CO‐FISH)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Combined Immunofluorescence and Telomere Fluorescence In Situ Hybridization (FISH) on Fixed Adherent Cells

  Materials
  • Adherent cells growing in culture
  • Appropriate tissue culture medium with serum (cell‐line specific)
  • Alcian blue stain (optional, see recipe)
  • Phosphate‐buffered saline (PBS; )
  • Pre‐extraction buffer (optional, see recipe)
  • 2% paraformaldehyde (see recipe)
  • KCM (see recipe)
  • ABDIL (see recipe)
  • 20 mg/ml DNase‐free RNase A stock
  • Normal goat serum
  • Primary antibodies
  • PBST (see recipe)
  • Goat‐derived, fluorophore‐conjugated secondary antibodies (Molecular Probes)
  • Graded ethanol series (70%, 90%, 100% v/v ethanol)
  • 0.3 μg/ml telomere PNA, Alexa Fluor 488–conjugated; see recipe)
  • DAPI (4′‐6‐diamidino‐2‐phenylindole)
  • PNA Wash A (see recipe)
  • PNA Wash B (see recipe)
  • Prolong Gold mounting medium (Life Technologies, cat. no. P10144)
  • Electron microscopy forceps (both sterile and unsterile)
  • 12‐mm round glass coverslips
  • 24‐well tissue culture plate
  • Bench top shaker
  • Menzel‐Glaser Superfrost slides
  • Humidified chamber
  • Heat block with thermostat control
  • Fluorescence microscope

Support Protocol 1: Combined Immunofluorescence and Telomere Fluorescent In Situ Hybridization (FISH) on Cytocentrifuged Chromosome Spreads (Metaphase‐TIF Assay)

  Additional Materials (also see protocol 1)
  • 10 μg/ml Colcemid (Karyomax) stock (Life Technologies, cat. no,. 15212‐012)
  • Hypotonic solution: 0.2% trisodium citrate/0.2% KCl (see recipe)
  • Phosphate‐buffered saline (PBS: ) containing 3.7% formaldehyde (see recipe)
  • α‐γH2A.X primary antibody (Millipore 05‐636, for metaphase‐TIF assay)
  • Cytocentrifuge clips, funnel, and filter papers (Thermo Fisher, cat. no. 3120110)
  • Cytocentrifuge (e.g., Cytospin 4 Cytocentrifuge from Thermo Fisher, cat. no. A78300003)
  • Coplin jars
  • Parafilm
  • 22 × 40–mm coverslips
  • DABCO antifade mountain medium (optional, see recipe)
  • Additional reagents and equipment for mammalian cell tissue culture including trypsinization and counting cells ( ; Phelan, )

Basic Protocol 2: In Vivo Detection of Telomeres in Archived Clinical Specimens

  Materials
  • Archived formalin‐fixed paraffin‐embedded clinical tissue specimens (4 to 5 μm sections)Xylene
  • Graded ethanol series (70%, 95%, and 100%)
  • 1% Tween 20 wash: 198 ml deionized distilled H 2O plus 2 ml Tween 20 (store at 4°C)
  • Antigen unmasking solution (Vector Laboratories)
  • PNA probes:
    • Telomere‐specific peptide nucleic acid (PNA) probe (CCCTAACCCTAACCCTAA with the N‐terminal covalently linked to Cy3)
    • Centromere‐specific PNA probe (ATTCGTTGGAAACGGGA with the N‐terminal covalently linked to Alexa Fluor 488)
  • PNA wash buffer (see recipe)
  • PBST (see recipe)
  • 500 ng/ml DAPI (4′‐6‐diamidino‐2‐phenylindole; Sigma)
  • Prolong antifade mounting medium (Molecular Probes)
  • Superfrost plus micro slides (VWR), or other positively charged slides
  • Slide warmer with thermostat control
  • Tissue‐Tek Slide Staining Set (VWR) or set of Coplin jars
  • Plastic slide holder
  • Kitchen steamer
  • Coverslips
  • Heat block with thermostat control
  • Humidified slide hybridization chamber (e.g., empty pipet tip box containing moist paper towel, protected from light)
  • Forceps
  • Fluorescence microscope equipped with appropriate fluorescence filter set

Support Protocol 2: In Vivo Detection of APBS in Archived Clinical Specimens

  Additional Materials (see protocol 1 and protocol 2; start after step 23)
  • Anti‐PML antibody (Dako, cat. no. PG‐M3)
  • Antibody dilution buffer (Ventana)
  • Anti‐mouse IgG fraction Alexa Fluor 488 (Molecular Probes)

Basic Protocol 3: Detection of Telomeric DNA Sequence on Mitotic Chromosome Spreads with Fluorescent In Situ Hybridization (FISH)

  Materials
  • Adherent cells growing in culture
  • 10 μg/ml Colcemid (Karyomax) stock (Life Technologies, cat. no,. 15212‐012)
  • Fixative (see recipe)
  • Hypotonic solution: 0.075 M KCl (dissolve 0.56 g KCl in 100 ml H 2O; prepare fresh and prewarm to 37°C)
  • 100% ethanol
  • Phosphate‐buffered saline (PBS; )
  • Phosphate‐buffered saline (PBS: ) containing 3.7% formaldehyde (see recipe)
  • 1 mg/ml pepsin (Sigma, cat. no. P7000)
  • Phosphate‐buffered saline (PBS) containing 250 μg/ml RNase A (see recipe)
  • Graded ethanol series (70%, 95%, and 100%)
  • 0.3 μg/ml telomere PNA probe, Alexa Fluor 488–conjugated, see recipe)
  • PNA Wash A (see recipe)
  • PNA Wash B (see recipe)
  • 500 ng/ml DAPI (4′‐6‐diamidino‐2‐phenylindole; Sigma)
  • Prolong Gold (Life Technologies, cat. no. P10144) or DABCO (see recipe) mounting medium
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Centrifuge
  • Coplin jars
  • Bright‐field microscope
  • Bench top shaker
  • Humidified chamber
  • 24 × 50 mm glass coverslips
  • Heat block with thermostatic control
  • Fluorescence microscope
  • Additional reagents and equipment for mammalian cell tissue culture including trypsinization and counting cells ( ; Phelan, )

Support Protocol 3: Detection of Telomere Sister Chromatid Exchange (T‐SCE) on Mitotic Chromosome Spreads by Chromosome Orientation FISH (CO‐FISH)

  Additional Materials (also see protocol 5)
  • 10 mM 3:1 BrdU/BrdC mixture (1000× stock; see recipe)
  • 20× SSC (see recipe)
  • 2× SSC with 2.5 mg/ml Hoechst 33258 (see recipe)
  • Exonuclease III (ExoIII) and corresponding buffer (NEB or Promega)
  • 70% deionized formamide:30% 2× SSC mixture
  • TexasRed‐G‐rich telomere PNA probe (see recipe)
  • Stratalinker 1800 UV irradiator (Stratagene)
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Figures

Videos

Literature Cited

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