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AOBPreview published online on September 28, 2005
Annals of Botany, doi:10.1093/aob/mci266
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© The Author 2005. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Received March 17, 2005
Revised May 25, 2005
Accepted August 10, 2005

Article

An Improved Method for Clearing and Staining Free-hand Sections and Whole-mount Samples*

ALEXANDER LUX 1*, SHIGENORI MORITA 2, JUN ABE 3, and KAORI ITO 2

1 Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Mlynská dolina B-2, SK--842 15 Bratislava, Slovak Republic
2 Field Production Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
3 AE-Bio, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan

* To whom correspondence should be addressed.
ALEXANDER LUX, E-mail: lux{at}fns.uniba.sk


  Abstract

• Background and Aims Free-hand sectioning of living plant tissues allows fast microscopic observation of internal structures. The aim of this study was to improve the quality of preparations from roots with suberized cell walls. A whole-mount procedure that enables visualization of exo- and endodermal cells along the root axis was also established.

• Methods Free-hand sections were cleared with lactic acid saturated with chloral hydrate, and observed with or without post-staining in toluidine blue O or aniline blue. Both white light and UV light were used for observation. Lactic acid was also used as a solvent for berberine, and fluorol yellow for clearing and staining the samples used for suberin observation. This procedure was also applied to whole-mount roots with suberized cell layers.

• Key Results Clearing of sections results in good image quality to observe the tissue structure and cell walls compared with non-cleared sections. The use of lactic acid as a solvent for fluorol yellow proved superior to previously used solvents such as polyethylene glycol-glycerol. Clearing and fluorescence staining of thin roots such as those of Arabidopsis thaliana were succesful for suberin visualization in endodermal cells within whole-mount roots. For thicker roots, such as those of maize, sorghum or tea, this procedure could be used for visualizing the exodermis in a longitudinal view. Clearing and staining of peeled maize root segments enabled observation of endodermal cell walls.

• Conclusions The clearing procedure using lactic acid improves the quality of images from free-hand sections and clearings. This method enhaces the study of plant root anatomy, in particular the histological development and changes of cell walls, when used in combination with fluorescence microscopy.

Keywords: Hand sections, Casparian bands, clearing, endodermis, exodermis, fluorescence microscopy.
* This work is dedicated to the 80th anniversary of the birth of the late Dr Mária Luxová.
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