Abstract
Imaging of fluorescent proteins in whole-mount tissue is a powerful tool to understand growth and developmental processes, not only in plants. With the advent of genetically encoded fluorescent reporters, which specifically label reproductive cells in Arabidopsis, deep tissue imaging has become increasingly important for the study of plant reproduction. To penetrate the surrounding layers of maternal tissue, however, the tissue has to be cleared by homogenizing the refractive index of the sample, often leading to inactivation of fluorescent proteins. 2,2′-thiodiethanol (TDE) has recently been introduced as a clearing agent that allows the imaging of fluorescent proteins in a cleared plant tissue. Here, we describe a simple protocol that combines TDE-based tissue clearing with cell wall staining to outline cells that enable deep tissue imaging in reproductive structures of Arabidopsis thaliana.
Key words
- Arabidopsis thaliana
- 2,2′-Thiodiethanol
- Optical clearing
- Deep tissue imaging
- Renaissance SR2200
- Embryogenesis
- Pollen tube
- Confocal microscopy
- 2-Photon microscopy
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Acknowledgments
Our research is funded by the German Science Foundation [Deutsche Forschungsgemeinschaft, DFG—SFB1101/B01 to M.B.] and the Max Planck Society. This protocol is based on previously published work [3, 9]. We like to thank Christian Liebig and Thomas Musielak for support and help in establishing the initial TDE-based clearing protocol.
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Slane, D., Bürgel, P., Bayer, M. (2017). Staining and Clearing of Arabidopsis Reproductive Tissue for Imaging of Fluorescent Proteins. In: Schmidt, A. (eds) Plant Germline Development. Methods in Molecular Biology, vol 1669. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7286-9_8
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DOI: https://doi.org/10.1007/978-1-4939-7286-9_8
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