ProtocolAn improved method for detection of apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain
Section snippets
Type of research
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Neuronal responses to axotomy [1].
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TUNEL technique to demonstrate apoptosis in dorsal root ganglia cells, in vitro and in vivo, after peripheral nerve damage [5].
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The TUNEL technique [6].
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Techniques for studying cell death alongside their relative merits and drawbacks [16].
Time required
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Tissue preparation: 2 h.
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TUNEL staining: 4 h.
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Hoechst staining/mounting: 30 min.
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Total protocol: 6 h and 30 min.
Materials
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P0 (day of birth, ∼7 g) Sprague–Dawley rats, male and female, were used for axotomy experiments. Adult male Sprague–Dawley rats (8–10 weeks, 180–200 g) were used for cell culture.
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Special equipment: Shandon 620 M cryostat.
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Chemicals and reagents: all chemicals were bought from Sigma, unless otherwise stated, and were of analytical grade.
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Poly-l-lysine, poly-dl-ornithine, laminin.
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Culture medium, consisting of Dulbecco's minimal Eagle's medium (DMEM) (Gibco BRL, Life Technologies, Paisley, UK),
Tissue preparation
Tissue (neonatal rat lumbosacral region, 17 day rat embryo (E17)) was dissected out, immediately frozen on dry ice and transferred to a −70°C freezer. Cryostat sections were cut (15 μm) in a Shandon 620 M cryostat, thaw mounted onto lysinated slides (poly-l-lysine, Sigma) and either used immediately or stored at −70°C. Adult rat dorsal root ganglia cells were grown on coverslips as previously described [3]. Before labelling, the coverslips were attached to slides using a small amount of
Results
TUNEL positive nuclei were identified in many systems of the embryonic rat, including small intestine, large intestine, liver and nervous tissue 6, 12. A large number of TUNEL positive nuclei were seen in the neonatal, post-injury dorsal root ganglia 1, 5. Likewise many TUNEL positive nuclei were seen in cultures of adult dorsal root ganglia. In all systems the background, non-specific TUNEL staining, was extremely low. Hoechst 33342 stained all nuclei in tissue sections and cultured cells,
Discussion
We have described a novel procedure in which the TUNEL technique is combined with subsequent staining with Hoechst 33342. The TUNEL technique labels fragmented DNA, the biochemical hallmark of apoptosis 2, 9, 10, 11, providing morphological information at a single cell level that verifies the production of DNA “ladders” on an agarose gel 4, 13, 14, 16. The TUNEL technique uses terminal deoxynucleotide transferase which increases its sensitivity over similar methods (in situ end-labelling
Quick procedure
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Sections were fixed in 4% w/v paraformaldehyde (10 min, room temperature (RT)) and washed in PBS (2×5 min, RT).
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The sections were then post-fixed in ethanol/acetic acid (2:1, 5 min, 4°C) and washed in PBS (2×5 min, RT), excess PBS was blotted off the slides.
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76 μl of equilibration buffer per slide was applied. Plastic coverslips were applied and the slides were placed in a humid chamber (5 min, RT). The coverslips were removed and excess liquid blotted off.
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54 μl of working strength TdT was
Essential literature references
Original papers: 2, 5, 6, 7.
Book: [15].
Reviews: 1, 12.
Acknowledgements
Work funded by Glaxo Wellcome R&D.
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