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

Mary‐Franciose Chesselet¹, Larami MacKenzie¹, Tuan Hoang¹

¹UCLA 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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Unit Introduction
Basic Protocol: 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: 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 3A 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

Figure 1.9.1

High-power bright-field photomicrograph from the substantia nigra pars compacta region. The labeling of DNA nick damage associated with individual neurons is illustrated. The clustering of silver grains over labeled neurons (black arrow) and the contrasting lack of silver grains over neighboring neurons (white arrow) indicate that a subset of neurons contains detectable DNA damage. Note that only cell nuclei are visible in this black and white photograph. Scale bar is 30 µm.

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Literature Cited

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.