Elsevier

Brain Research Protocols

Volume 2, Issue 2, January 1998, Pages 160-164
Brain Research Protocols

Protocol
An improved method for detection of apoptosis in tissue sections and cell culture, using the TUNEL technique combined with Hoechst stain

https://doi.org/10.1016/S1385-299X(97)00032-9Get rights and content

Abstract

We describe a novel procedure for combining a fluorescent variant of the TUNEL technique with Hoechst 33342 stain (bis-benzimide) to identify apoptosis in tissue sections and cell culture. The biochemical hallmark of apoptosis is internucleosomal DNA cleavage, which gives rise to oligonucleosome-sized fragments (multiples of approximately 180 bp) that can be directly visualised by labelling with biotinylated or digoxygenin-conjugated nucleosides in a reaction that employs terminal deoxynucleotide transferase (TUNEL). TUNEL and Hoechst 33342 have been used separately to identify apoptosis. TUNEL specifically labels dying cells, yet a low background makes comparison of labelled cells with surrounding normal cells difficult and causes disorientation in tissue sections. Hoechst 33342 binds all DNA therefore staining all nuclear material, allowing identification of apoptotic nuclei, but the analysis is laborious. Combining the two fluorescent labels allows the initial recognition of apoptotic cells using the TUNEL technique then, by simply changing the filter, the TUNEL positive nuclei can be compared to surrounding normal nuclei to assess changes in morphology and size. Hoechst 33342 acts as a counterstain, allowing identification of anatomical structures, and permits quantitative comparison between TUNEL positive versus normal cells. We have evaluated the technique using sections of rat embryo, post-axotomy neonatal dorsal root ganglia and adult dorsal root ganglia cell culture.

Section snippets

Type of research

  • Neuronal responses to axotomy [1].

  • TUNEL technique to demonstrate apoptosis in dorsal root ganglia cells, in vitro and in vivo, after peripheral nerve damage [5].

  • The TUNEL technique [6].

  • Techniques for studying cell death alongside their relative merits and drawbacks [16].

Time required

  • Tissue preparation: 2 h.

  • TUNEL staining: 4 h.

  • Hoechst staining/mounting: 30 min.

  • Total protocol: 6 h and 30 min.

Materials

  • 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.

  • Special equipment: Shandon 620 M cryostat.

  • Chemicals and reagents: all chemicals were bought from Sigma, unless otherwise stated, and were of analytical grade.

  • Poly-l-lysine, poly-dl-ornithine, laminin.

  • 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

  • Sections were fixed in 4% w/v paraformaldehyde (10 min, room temperature (RT)) and washed in PBS (2×5 min, RT).

  • 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.

  • 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.

  • 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.

References (17)

There are more references available in the full text version of this article.

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