Detection of DNA Damage in Tissue Sections by In Situ Nick Translation

Mary‐Franciose Chesselet1, Larami MacKenzie1, Tuan Hoang1

1 UCLA School of Medicine, Los Angeles, California
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.9
DOI:  10.1002/0471142301.ns0109s16
Online Posting Date:  November, 2001
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Abstract

The technique of in situ nick translation (ISNT) is used to detect DNA strand breaks in tissue sections at the cellular level with great sensitivity. In fact, ISNT can be used to detect DNA damage in a single cell, which is particularly useful to assess programmed cell death during development. One crucial advantage of ISNT is the anatomical resolution that permits a detailed topographical analysis of DNA damage. This can be useful to identify cells that are more vulnerable to an experimental insult. Furthermore, cells with DNA damage can be identified morphologically with this method. This can be of interest to determine whether cells that exhibit DNA damage already exhibit clear features of dying cells or are still relatively intact morphologically. This can be useful to identify the mode of cell death involved. This unit provides a protocol that describes tissue preparation, in situ nick translation and emulsion autoradiography.

     
 
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Table of Contents

  • Basic Protocol 1: Detection of DNA Damage in Tissue Sections by In Situ Nick Translation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Detection of DNA Damage in Tissue Sections by In Situ Nick Translation

  Materials
  • Brain tissue sample (unit 1.1), fresh frozen or perfused
  • 3% paraformaldehyde (unit 1.1) in 0.1 M PBS, for fresh‐frozen tissue
  • 0.1 M PBS: dilute from sterile 0.4 M PBS/3.6% saline stock (see recipe) up to one day ahead of use, keep at 4°C
  • 2× SSC: dilute from sterile 20× SSC ( appendix 2A) on day of use
  • 50/50 (v/v) formamide/2× SSC (make fresh on day of use), warmed to 52°C before use
  • Acetic anhydride
  • 0.1 M TEA (see recipe)
  • ISNT buffer (see recipe; add 10 mM 2‐mercaptoethanol just before use)
  • Nick mix (see recipe)
  • DNA polymerase I
  • 50 mM Tris⋅Cl, pH 7.5 ( appendix 2A), ice‐cold
  • 70%, 80%, 95%, and 100% ethanol
  • Glass hybridization jars (Fisher)
  • Gelatin‐subbed glass slides (unit 1.1)
  • Slide boxes and desiccant (Fisher)
  • 52°C water bath
  • Glass coverslips, 24 × 30–mm (e.g., Fisher)
  • Slide drying tray
  • Humid chamber: sealable plastic container (e.g., Tupperware) containing a water‐saturated paper insert
  • Double‐faced tape
  • Additional materials for cryostat sectioning of tissue (unit 1.1), film autoradiography (CPMB APPENDIX and appendix 1A in this manual), emulsion autoradiography (unit 1.1), and Nissl staining (unit 1.1)
NOTE: Use sterile water and sterile containers for all solutions.
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Figures

Videos

Literature Cited

   Bordelon, Y.M., MacKenzie, L., and Chesselet, M.‐F. 1999. Morphology and compartmental location of cells exhibiting DNA damage after quinolinic acid injections into rat striatum. J. Comp. Neurol. 412:38‐50.
   Chesselet, M.‐F. 1996. Quantitative analysis and interpretation of data for in situ hybridization at the cellular level. In Situ Hybridization Techniques for the Brain (Z. Henderson, ed.) pp. 141‐149. IBRO Handbook Series: Methods in the Neurosciences. International Brain Research Organization, Paris.
   Didier, M., Bursztajn, S., Adamec, E., Passani, L., Nixon, R.A., Coyle, J.T., Wey, J.Y., and Berman, S.A. 1996. DNA strand breaks induced by sustained glutamate excitotoxicity in primary neuronal cultures. J. Neurosci. 16:2238‐2250.
   Iseki, S. and Mori, T. 1985. Histochemical detection of DNA strand scission in mammalian cells by in situ nick translation. Cell Biol. Int. Rep. 9:471‐477.
Key References
   Bordelon et al., 1999. See above.
  Describes the use of the technique for establishing the time course of DNA damage and the precise location of cells with DNA damage.
   Chesselet, 1996. See above.
  Discusses general principles and precautions for quantitative autoradiography.
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