A protocol for combining fluorescent proteins with histological stains for diverse cell wall components.

Details

Serval ID
serval:BIB_F18CEAFA629F
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A protocol for combining fluorescent proteins with histological stains for diverse cell wall components.
Journal
The Plant Journal
Author(s)
Ursache R., Andersen T.G., Marhavý P., Geldner N.
ISSN
1365-313X (Electronic)
ISSN-L
0960-7412
Publication state
Published
Issued date
2018
Peer-reviewed
Oui
Volume
93
Number
2
Pages
399-412
Language
english
Abstract
Higher plant function is contingent upon the complex three-dimensional (3D) architecture of plant tissues, yet severe light scattering renders deep, 3D tissue imaging very problematic. Although efforts to 'clear' tissues have been ongoing for over a century, many innovations have been made in recent years. Among them, a protocol called ClearSee efficiently clears tissues and diminishes chlorophyll autofluorescence while maintaining fluorescent proteins - thereby allowing analysis of gene expression and protein localisation in cleared samples. To further increase the usefulness of this protocol, we have developed a ClearSee-based toolbox in which a number of classical histological stains for lignin, suberin and other cell wall components can be used in conjunction with fluorescent reporter lines. We found that a number of classical dyes are highly soluble in ClearSee solution, allowing the old staining protocols to be enormously simplified; these additionally have been unsuitable for co-visualisation with fluorescent markers due to harsh fixation and clearing. Consecutive staining with several dyes allows 3D co-visualisation of distinct cell wall modifications with fluorescent proteins - used as transcriptional reporters or protein localisation tools - deep within tissues. Moreover, the protocol is easily applied on hand sections of different organs. In combination with confocal microscopy, this improves image quality while decreasing the time and cost of embedding/sectioning. It thus provides a low-cost, efficient method for studying thick plant tissues which are usually cumbersome to visualise. Our ClearSee-adapted protocols significantly improve and speed up anatomical and developmental investigations in numerous plant species, and we hope they will contribute to new discoveries in many areas of plant research.

Keywords
ClearSee, cell wall staining, cellulose, cutin, fluorescent proteins, hand sections, lignin, suberin, technical advance
Pubmed
Web of science
Create date
30/11/2017 19:21
Last modification date
20/08/2019 16:19
Usage data