Abstract
Visualization of nuclei in S-phase cells in tissues is important for not only cell cycle research but also developmental research because morphogenesis is usually achieved by a combination of cell proliferation and cell expansion. Recently, DNA labeling with 5-ethynyl-2′-deoxyuridine (EdU), which is an analog of thymidine, has been used to visualize nuclei in S-phase cells to assess the activity of cell proliferation during development of plants. EdU is efficiently incorporated into newly synthesized DNA, and detection of EdU is based on the covalent reaction between EdU and Alexa Fluor® dye, which is one of useful fluorescent dyes; this allows us to use mild conditions for the assay without any DNA denaturation. This method could be easily applicable, and, indeed, has been used for various model and non-model plant species. Here, we have described a protocol developed for the detection of nuclei in S-phase cells in leaves.
Keywords: Cell division, Cell proliferation, 5-ethynyl-2′-deoxyuridine (EdU), Rorippa aquatica
Materials and Reagents
Equipment
Procedure
Representative data
For representative data, please see the papers of Ichihashi et al. (2014) and Nakayama et al. (2014).
Notes
Recipes
Acknowledgments
The protocol was modified from Kotogány et al. (2010). We thank Ms. Rumi Amano for preparing the figures. This research was partially supported by Grants-in-Aid from the Japan Society for the Promotion of Science (JSPS) (KAKENHI Grant Numbers 22870031, 24247007, 24770047 and 25113002) and The Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan to S. K. and BIO-NEXT project from Okazaki Institute for Integrative Bioscience to K. K. and H. T. and by a Research Fellowship from JSPS to H. N..
References
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