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Improved Methods for Clearing and Staining of Plant Samples

Abstract

The free-hand sectioning represents a suitable method for fast microscopic observation and microphotography. The aim of this protocol is to improve the quality of preparations with emphasis on roots and visualization of suberized and lignified cell walls. Free-hand sections were cleared with lactic acid or lactic acid saturated with chloral hydrate, and observed with or without a post-staining step in toluidine blue O or aniline blue. For suberin detection in fluorescence microscopy, lactic acid was used as a solvent for berberine and fluorol yellow. This method was applied to both hand sections and whole-mount thin roots with suberized cell layers. Cleared samples were also exposed to phloroglucinol and HCl (hydrochloric acid) for detection of lignin. Clearing provides superior image quality for observation compared to non-cleared samples.

Keywords

  • Casparian bands
  • Clearing
  • Endodermis
  • Exodermis
  • Fluorescence microscopy
  • Hand sections
  • Lignin
  • Suberin
  • Whole-mount samples

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Acknowledgments

The work was supported by Slovak Research and Development Agency under the contract Nr. APVV-0140-10, APVV SK-CN-0016-12, APVV SK-FR-2013-0029, VEGA 1/0817/12 and was a part of COST FA 0905 Action.

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Correspondence to Alexander Lux .

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Lux, A., Vaculík, M., Kováč, J. (2015). Improved Methods for Clearing and Staining of Plant Samples. In: Yeung, E., Stasolla, C., Sumner, M., Huang, B. (eds) Plant Microtechniques and Protocols. Springer, Cham. https://doi.org/10.1007/978-3-319-19944-3_10

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