An improved method for clearing and staining free-hand sections and whole-mount samples
An improved method for clearing and staining free-hand sections and whole-mount samples
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DOI：
10.1093/aob/mci266
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发表时间：
2005-11-01
期刊：
影响因子：
4.2
通讯作者：
Ito, K
中科院分区：
文献类型：
--
作者：
Lux, A;Morita, S;Ito, K
背景和目标活体植物组织的徒手切片可以快速地用显微镜观察内部结构。本研究的目的是提高具有木栓化细胞壁的根制剂的质量。还建立了一种能够沿着根轴可视化外胚层和内胚层细胞的整体安装程序。方法用水合氯醛饱和的乳酸透明化徒手切片，并在有或没有甲苯胺蓝O或苯胺蓝后染色的情况下进行观察。使用白光和紫外光进行观察。乳酸也用作小檗碱的溶剂，氟酚黄用于对用于木栓质观察的样品进行透明和染色。该程序也适用于具有木栓化细胞层的整体根。 主要结果 与未透明切片相比，透明切片可产生良好的图像质量，可观察组织结构和细胞壁。事实证明，使用乳酸作为氟酚黄溶剂优于以前使用的溶剂，例如聚乙二醇-甘油。对拟南芥等细根进行透明化和荧光染色，成功实现了整个根内内胚层细胞中木栓质的可视化。对于较粗的根，例如玉米、高粱或茶的根，该程序可用于在纵向视图中可视化外皮。对去皮的玉米根段进行透明化和染色可以观察内胚层细胞壁。结论使用乳酸的透明化程序提高了徒手切片和透明化的图像质量。当与荧光显微镜结合使用时，该方法增强了植物根解剖学的研究，特别是细胞壁的组织学发育和变化。
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 celllayers.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.