Abstract
Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) is a method for detecting DNA fragmentation by labelling the 3' terminal end of nucleic acids. This method can be used both in animal and plant tissues. In animal tissues, the use of Proteinase K is sufficient for permeabilizing the cells and to obtain optimal labelling, but in plant tissues, the presence of the cell wall, does not allow proper labelling. For this reason, we carried out several modifications to the original TUNEL protocol (ApoAlert® DNA Fragmentation Assay Kit, Clontech) to obtain an optimal labelling. These modifications were additional treatments with cellulase, Triton X-100 and Proteinase K. Also, we describe the optimization of the positive controls by adjusting the units of DNase used. The PI concentration for counterstaining has been also specifically adjusted to avoid excessive background noise and hence to correctly observe both labeled and unlabelled nuclei. This work also describes an additional protocol to collect, store and include samples (specifically stigmatic arms) in such a way that they do not interfere with the TUNEL labelling.
Keywords: Programmed Cell Death, TUNEL assay, DNA fragmentation assay, Kiwifruit, Plant reproductive biology
Materials and Reagents
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Representative data
This improved protocol for TUNEL assay of DNA fragmentation in kiwifruit tissues relies on the use of an improved permeabilization of tissues by treatments with cellulase, Triton X-100, and adjusted Proteinase K, increased DNase I units (1,500 U/ml) for the generation of positive controls, and optimization of counterstaining with reduced IP concentration (0.5 μg/ml). These modifications allowed us to clearly visualize the differences between negative and positive nuclei for TUNEL (Figure 3A-C), in contrast with the results obtained using the original protocol of the ApoAlert® DNA Fragmentation Assay Kit, which was not specifically designed for plant tissues (Figure 3D-F). In the Figure 3A, TUNEL-positive nuclei can be observed as they are showing bright green fluorescence, while in the Figure 3B all present nuclei are stained in red. Finally, in the Figure 3C, all present nuclei can be observed, but those being TUNEL positive are stained in yellow whereas those TUNEL negative are stained in light red or orange. On the contrary, with the ApoAlert® kit (Figure 3D-F) since no TUNEL-positive nuclei can be observed (Figure 3D), all present nuclei can be observed in red (Figure 3E) or after counterstaining in orange (Figure 3F). Figure 3. Comparison of TUNEL assays in positive controls of kiwifruit tissues. A-C. Positive control sections of kiwifruit stigmatic arms stained using the improved TUNEL protocol described here. D-F. Positive control sections of kiwifruit stigmatic arms stained using the ApoAlert® DNA Fragmentation Assay Kit (Clontech). A, D. Fluorescence detected at 488 nm for the observation of the TUNEL stained nuclei (arrows). B, E. Fluorescence detected at 561 nm for the observation of the IP stained nuclei (arrows). C, F. Counterstaining by simultaneous fluorescence detection at both wavelengths (488 and 561 nm) of stained nuclei with TUNEL and IP (arrows).
Recipes
Acknowledgments
This research was supported by the Xunta de Galicia (Regional Government of Galicia, Spain; project PGIDIT 04RAG291002PR). We thank Kiwi Atlántico S. A. for providing the plant material. We thank M. S. Costa for her expert photographic assistance, and B. Lueiro for her technical assistance. This is a contribution of the Interuniversity Research Group in Biotechnology and Reproductive Biology of Woody Plants (BioVitAc Research group, code 09IDI1705).
References
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