Transverse Sectioning of Arabidopsis thaliana Leaves Using Resin Embedding
树脂包埋制备拟南芥横切片
(*contributed equally to this work) 发布: 2015年09月20日第5卷第18期 DOI: 10.21769/BioProtoc.1592 浏览次数: 14154
评审: Maria SinetovaMoritz BomerRumen Ivanov
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参见作者原研究论文
The authors used this protocol in:
Dec 2014
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Abstract
The leaf is the major functional part of the shoot performing the bulk of photosynthetic activity. Its development is relatively plastic allowing the plant to adapt to environmental changes by modifying leaf size and anatomy. Moreover, a leaf is made up of various distinct cell layers, each having specialized functions. To understand functional adaptation and the development of the leaf it is essential to obtain cross sections throughout leaf development and at maturity (Kalve et al., 2014). Here, we describe a protocol for transverse sectioning of Arabidopsis thaliana leaves using resin embedding. This protocol provides a reliable platform to yield high quality images of cross sections allowing study of development of various tissue layers across the transversal axis of the leaf. As this method is an adaptation of the protocol developed for the Arabidopsis root tip by Beeckman and Viane (1999) and De Smet et al. (2004), it can easily be modified to accommodate other organs and species.
Keywords: Microscopy (显微镜)
Arabidopsis thaliana (拟南芥)
Leaves (叶)
Microtomy (切片)
Materials and Reagents
- Arabidopsis thaliana
- Acetic Acid, Glacial (Certified ACS), Fisher Chemical (Fisher Scientific, catalog number: A38-212 )
- Formaldehyde solution (Sigma-Aldrich catalog, number: 47629 )
- Ethanol (VWR International, catalog number: 83813360 )
- Acetic anhydride (Sigma-Aldrich, catalog number: 320102 )
- TWEEN® 20 (Sigma-Aldrich, catalog number: P9416 )
- NaH2PO4.H2O (Fisher Scientific, catalog number: AC2717500190 )
- Technovit 7100 with hardener I and II (Heraeus Kulzer GmbH, catalog number: 64709003 )
- Toluidine blue (EMD Millipore Corporation, catalog number: 1159300025 )
- DPX mounting medium (a mixture of Distyrene, a Plasticizer and Xylene) (Sigma- Aldrich, catalog number: M1289-10 ML )
- Object slides (superfrost plus adhesion slides) (Thermo Fisher Scientific, catalog number: 10143352 )
- Glass microtome knives (SPI Easy-cut Ultra/Glass, catalog number: 07668-BA )
- Cover slips
- Microcentrifuge tubes, PCR tubes or molds (Electron microscopy sciences, catalog number: 70900 )
- Phosphate buffer (see Recipes)
- Technovit solution (see Recipes)
- Technovit solution with hardener-II (see Recipes)
Equipment
- Vacuum pump (Welch USA- BRS, catalog number: 2522C-02 )
- Vacuum jar
- Tweezers
- Heated plate (Bekso, model: TH3N )
- Rotary microtome (Leica Reichert-Jung, catalog number: 2040 )
- Bright field microscope (ZEISS, model: Axio Scope A1 ) fitted with a digital camera (ZEISS, model: AxioCamCm1 )
Procedure
文章信息
版权信息
© 2015 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Kalve, S., Saini, K., Vissenberg, K., Beeckman, T. and Beemster, G. T. S. (2015). Transverse Sectioning of Arabidopsis thaliana Leaves Using Resin Embedding . Bio-protocol 5(18): e1592. DOI: 10.21769/BioProtoc.1592.
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分类
植物科学 > 植物细胞生物学 > 细胞成像
植物科学 > 植物细胞生物学 > 组织分析
细胞生物学 > 细胞成像 > 固定组织成像
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