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Transverse Sectioning of Arabidopsis thaliana Leaves Using Resin Embedding
树脂包埋制备拟南芥横切片   

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Journal of Experimental Botany
Dec 2014

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

  1. Arabidopsis thaliana
  2. Acetic Acid, Glacial (Certified ACS), Fisher Chemical (Fisher Scientific, catalog number: A38-212 )
  3. Formaldehyde solution (Sigma-Aldrich catalog, number: 47629 )
  4. Ethanol (VWR International, catalog number: 83813360 )
  5. Acetic anhydride (Sigma-Aldrich, catalog number: 320102 )
  6. TWEEN® 20 (Sigma-Aldrich, catalog number: P9416 )
  7. NaH2PO4.H2O (Fisher Scientific, catalog number: AC2717500190 )
  8. Technovit 7100 with hardener I and II (Heraeus Kulzer GmbH, catalog number: 64709003 )
  9. Toluidine blue (EMD Millipore Corporation, catalog number: 1159300025 )
  10. DPX mounting medium (a mixture of Distyrene, a Plasticizer and Xylene) (Sigma- Aldrich, catalog number: M1289-10 ML )
  11. Object slides (superfrost plus adhesion slides) (Thermo Fisher Scientific, catalog number: 10143352 )
  12. Glass microtome knives (SPI Easy-cut Ultra/Glass, catalog number: 07668-BA )
  13. Cover slips
  14. Microcentrifuge tubes, PCR tubes or molds (Electron microscopy sciences, catalog number: 70900 )
  15. Phosphate buffer (see Recipes)
  16. Technovit solution (see Recipes)
  17. Technovit solution with hardener-II (see Recipes)

Equipment

  1. Vacuum pump (Welch USA- BRS, catalog number: 2522C-02 )
  2. Vacuum jar
  3. Tweezers
  4. Heated plate (Bekso, model: TH3N )
  5. Rotary microtome (Leica Reichert-Jung, catalog number: 2040 )
  6. Bright field microscope (ZEISS, model: Axio Scope A1 ) fitted with a digital camera (ZEISS, model: AxioCamCm1 )

Procedure

  1. Fixation: Dissect and fix whole Arabidopsis leaf blades under a vacuum in a solution of 5% acetic acid, 5% formaldehyde and 50% ethanol for an hour. Alternatively, ethanol:acetic anhydride (3:1) with a drop of Tween-20 can be used for fixation. Fixation is preferentially performed in glass vials to allow visualization of the leaves and multiple replicate leaves from a single stage/treatment can be treated in the same volume. The samples should sink as this is indicative of a good penetration of the fixative. Store the leaves overnight in the same fixation solution at 4 °C.
  2. Rinse: Rinse the fixed leaf samples with phosphate buffer (pH 7.2).
  3. Dehydration: Dehydrate the tissues by sequential incubations (Note 1) in a volume of ethanol sufficient to submerge freely specimens floating, without being constricted by the side walls of the container:
    - 30% ethanol for 4 h
    - 50% ethanol for 4 h
    - 70% ethanol for 4 h, overnight or sample can be stored at 4 °C for a maximum of 2 weeks
    - 80% ethanol for 4 h
    - 90% ethanol for 4 h
    - 95% ethanol for 4 h or overnight at 4 °C
    - 98% ethanol for 1 h
    - 99% ethanol for 1 h
    - 100% ethanol for 1 h. Repeat this step one more time.
  4. Pre-infiltration: To replace the ethanol with Technovit 7100 resin (Figure 1a) perform following steps (Note 2):
    - 30% Technovit solution and 70% ethanol (consisting of 100% ethanol) for 15 h
    - 50% Technovit solution and 50% ethanol (consisting of 100% ethanol) for 15 h
    - 70% Technovit solution and 30% ethanol (consisting of 100% ethanol) for 15 h
  5. Infiltration: Replace the above solution with 100% Technovit solution and incubate for 15 h at room temperature.
  6. Polymerization: Place the leaf samples in molds (Figure 1b-f) or microcentrifuge tubes (Figure 1g-h) and add Technovit solution with hardener-II. Let it polymerize overnight at 37 °C in a closed environment i.e. cover the molds or cap microcentrifuge tubes to avoid evaporation (Note 3). Samples can be embedded horizontally in the molds as shown in Figure 1e or flat molds can be made between 2 overhead projection transparencies (Beeckman and Viane, 1999), which are then re-embedded vertically in microcentrifuge tubes.


    Figure 1. Subsequent steps of the protocol. a. overview of the work space; b-c. preparing and adding Technovit solution with hardener–II to the molds; d-e. placing the sample at the desired orientation; f. polymerized resin block containing sample (arrow); g-h. alternatively PCR tubes can be used as molds; i. resin block attached to microtome sample holder; j. samples on a slide placed on the heated plate; k. transverse, stained section of leaf visualized using a bright field microscope.

  7. Position the polymerized samples horizontally (Figure 1i) in the microtome to obtain the transverse sections. Make the sections of required thickness (in this case 2 μm) with a glass knife on the rotary microtome.
  8. Take the piece of section with tweezers and place it into a drop of water on an object slide.
  9. Place the object slide on the heated plate at 30-40 °C and let it dry (Figure 1j).
  10. Staining: Stain the sections on the object slide with a 0.05% (w/v) solution of toluidine blue for 10 min and rinse with distilled water for 1 min.
  11. Dry the sections on the object slide at 30-40 °C on the heated plate and mount the cover slip using DEPEX as a mounting agent.
  12. Take pictures of the sections at 20x or 40x magnification with a bright field microscope fitted with a digital camera (Figure 1k).

Notes

  1. All the sequential incubation steps should be performed without changing the container and removing the solutions between incubation steps by pipette to avoid tissue damage.
  2. Samples can be pre-stained by neutral red or toluidine blue just before embedding to aid in visualization, for desired orientation.
  3. Technovit resin embedding is compatible with a broad range of stainings including immunolocalisation, enzyme histochemistry, in situ hybridization, and a range of classical histological stains (Willbold and Witte, 2010).

Recipes

  1. Phosphate buffer
    Add 13.799 g NaH2PO4.H2O in 1 L water
    Adjust the pH to 7.2
  2. Technovit solution
    Add 1 g hardener-I in 100 ml Technovit 7100, protect it from light
    It can be stored at 4 °C for few weeks
  3. Technovit solution with hardener-II
    Add 1 ml hardener-II in 15 ml Technovit solution, mix it by stirring and keep on ice to avoid polymerization
    Make the fresh solution and use it immediately

Acknowledgments

We would like to thank Maria Njo for technical support. This work was supported by a concerted research activity (GOA) research grant, ‘A Systems Biology Approach of Leaf Morphogenesis’ from the research council of the University of Antwerp and an Interuniversity Attraction Poles project (IAP7/29, MARS) from the Belgian Federal Science Policy Office (BELSPO). Shweta Kalve was funded by a training grant from the Department of Science and Education of the Flemish Government. The authors acknowledge the financial support of the Research Foundation-Flanders (FWO-Vlaanderen).

References

  1. Beeckman, T. and Viane, R. (2000). Embedding thin plant specimens for oriented sectioning. Biotech Histochem 75(1): 23-26.
  2. De Smet, I., Chaerle, P., Vanneste, S., De Rycke, R., Inze, D. and Beeckman, T. (2004). An easy and versatile embedding method for transverse sections. J Microsc 213 (Pt 1): 76-80.
  3. Kalve, S., Fotschki, J., Beeckman, T., Vissenberg, K. and Beemster, G. T. (2014). Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves. J Exp Bot 65(22): 6385-6397.
  4. Willbold, E. and Witte, F. (2010). Histology and research at the hard tissue-implant interface using Technovit 9100 New embedding technique. Acta Biomater 6(11): 4447-4455.

简介

叶是进行大量光合活性的枝条的主要功能部分。其发育是相对塑性的,允许植物通过改变叶的大小和解剖学适应环境变化。此外,叶片由各自不同的细胞层组成,每个细胞层具有专门的功能。为了理解功能适应和叶的发育,必须在叶发育和成熟期间获得横截面(Kalve等人,2014)。在这里,我们描述了使用树脂嵌入的拟南芥叶片的横切片的方案。这个协议提供了一个可靠的平台,产生高质量的横截面图像,允许研究横跨叶片横向轴线的各种组织层的发展。因为该方法是Beeckman和Viane(1999)和De Smet等人(2004)为拟南芥根尖所开发的方案的改编,所以它可以容易地修改以适应其他器官和物种。

关键字:显微镜, 拟南芥, 叶, 切片

材料和试剂

  1. 拟南芥
  2. 乙酸,冰川(Certified ACS),Fisher Chemical(Fisher Scientific,目录号:A38-212)
  3. 甲醛溶液(Sigma-Aldrich目录,编号:47629)
  4. 乙醇(VWR International,目录号:83813360)
  5. 乙酸酐(Sigma-Aldrich,目录号:320102)
  6. TWEEN 20(Sigma-Aldrich,目录号:P9416)
  7. (Fisher Scientific,目录号:AC2717500190),其中所述化合物具有以下结构:其中R 1,R 2,R 3,R 4,
  8. 具有硬化剂I和II(Heraeus Kulzer GmbH,目录号:64709003)的Technovit 7100
  9. 甲苯胺蓝(EMD Millipore Corporation,目录号:1159300025)
  10. DPX安装介质(苯乙烯,增塑剂和二甲苯的混合物)(Sigma-Aldrich,目录号:M1289-10ML)
  11. 对象载玻片(superfrost plus adhesion slides)(Thermo Fisher Scientific,目录号:10143352)
  12. 玻璃切片刀(SPI Easy-cut Ultra/Glass,目录号:07668-BA)
  13. 封面
  14. 微量离心管,PCR管或模具(电子显微镜科学,目录号:70900)
  15. 磷酸盐缓冲液(参见配方)
  16. Technovit解决方案(参见配方)
  17. 具有硬化剂II的Technovit溶液(参见配方)

设备

  1. 真空泵(Welch USA-BRS,目录号:2522C-02)
  2. 真空瓶
  3. 镊子
  4. 加热板(Bekso,型号:TH3N)
  5. 旋转切片机(Leica Reichert-Jung,目录号:2040)
  6. 装配有数字照相机(ZEISS,型号:AxioCamCm1)的明视场显微镜(ZEISS,型号:Axio Scope A1)

程序

  1. 固定:在5%乙酸,5%甲醛和50%乙醇的溶液中在真空下解剖和固定整个拟南芥叶片1小时。或者,具有一滴Tween-20的乙醇:乙酸酐(3:1)可用于固定。固定优选在玻璃小瓶中进行以允许叶的可视化,并且来自单个阶段/处理的多个重复叶可以在相同体积中处理。样品应该下沉,因为这指示固定剂的良好渗透。将叶子在4℃下在相同的固定溶液中存储过夜
  2. 冲洗:用磷酸盐缓冲液(pH 7.2)冲洗固定叶样品
  3. 脱水:在一定体积的乙醇中通过连续孵育(注1)使组织脱水,足以浸没自由浮动的标本,而不会被容器的侧壁收缩:
    - 30%乙醇,持续4小时
    - 50%乙醇,持续4小时
    - 70%乙醇4小时,过夜或样品可以在4°C下存储最多2周
    - 80%乙醇4小时
    - 90%乙醇4小时
    - 95%乙醇4小时或在4℃过夜
    - 98%乙醇1小时
    - 99%乙醇1小时
    - 100%乙醇1小时。重复此步骤一次。
  4. 预浸渍:要用Technovit 7100树脂(图1a)替换乙醇,请执行以下步骤(注2):
    - 30%Technovit溶液和70%乙醇(由100%乙醇组成)15小时 - 50%Technovit溶液和50%乙醇(由100%乙醇组成)15小时 -70%Technovit溶液和30%乙醇(由100%乙醇组成)15小时
  5. 渗透:用100%Technovit溶液替换上述溶液,并在室温下孵育15小时。
  6. 聚合:将叶片样品置于模具(图1b-f)或微量离心管(图1g-h)中,并加入具有硬化剂II的Technovit溶液。让它在37℃下在封闭的环境中聚合过夜,即, 覆盖模具或盖微量离心管以避免蒸发(注3)。样品可以水平嵌入模具中,如图1e所示,或者平面模具可以在2个投影投影透明片(Beeckman和Viane,1999)之间制成,然后将其垂直嵌入微量离心管中。


    图1.协议的后续步骤。工作空间概述;公元前。制备和添加具有硬化剂II的Technovit溶液至模具; d-e。将样品放置在期望的取向; F。聚合树脂块含有样品(箭头); g-h。或者PCR管可用作模具;一世。树脂块连接到切片机样品架上; j。放在加热板上的载玻片上的样品; k。横向,染色的叶片使用明视场显微镜可视化
  7. 将聚合的样品水平放置(图1i)在切片机中以获得横切片。使用旋转切片机上的玻璃刀切割所需厚度的切片(在这种情况下为2μm)
  8. 用镊子取出一个部分,放入一个水滴在物体幻灯片上。
  9. 将物体载玻片放在加热板上,温度为30-40°C,使其干燥(图1j)
  10. 染色:用0.05%(w/v)甲苯胺蓝溶液将载物片上的切片染色10分钟,并用蒸馏水冲洗1分钟。
  11. 在加热板上在30-40℃下干燥物体载片上的部分,并使用DEPEX作为固定剂安装盖玻片。
  12. 使用配有数码相机的明场显微镜以20倍或40倍放大倍数拍摄切片的照片(图1k)。

笔记

  1. 所有连续的孵育步骤应该在不改变容器和移除培养步骤之间的溶液通过移液器进行,以避免组织损伤。
  2. 样品可以在包埋之前用中性红或甲苯胺蓝预染色,以帮助可视化,达到所需的取向
  3. Technovit树脂嵌入与广泛的染色,包括免疫定位,酶组织化学,原位杂交和一系列经典的组织学染色(Willbold和Witte,2010)兼容。

食谱

  1. 磷酸盐缓冲液
    在1L水中加入13.799g NaH 2 PO 4 subO 2 .H 2 H 2 O在
    将pH调节至7.2
  2. Technovit溶剂
    在100毫升Technovit 7100中加入1克硬化剂I,避免光线照射
    它可以在4℃保存几个星期
  3. 具有硬化剂II的Technovit溶液
    在15ml Technovit溶液中加入1ml硬化剂II,搅拌混合并保持在冰上避免聚合
    制作新鲜溶液并立即使用

致谢

我们要感谢Maria Njo的技术支持。 这项工作得到了安特卫普大学研究委员会和比利时联邦大学吸引力项目(IAP7/29,MARS)的一项协调研究活动(GOA)研究资助,"A Systems Biology Approach of Leaf Morphogenesis" 科学政策办公室(BELSPO)。 Shweta Kalve由佛兰芒政府科学和教育部的培训资助。 作者承认研究基金会佛兰德斯(FWO-Vlaanderen)的财政支持。

参考文献

  1. Beeckman,T。和Viane,R。(2000)。 嵌入薄植物样本用于定向切片。 生物技术Histochem 75(1):23-26
  2. De Smet,I.,Chaerle,P.,Vanneste,S.,De Rycke,R.,Inze,D.and Beeckman,T。(2004)。 横断面的简单而多用的嵌入方法 J Microsc < em> 213(Pt 1):76-80
  3. Kalve,S.,Fotschki,J.,Beeckman,T.,Vissenberg,K.and Beemster,G.T。(2014)。 拟南芥的整个发育过程中细胞分裂和扩增的三维模式叶。 65(22):6385-6397。
  4. Willbold,E。和Witte,F。(2010)。 使用Technovit 9100新嵌入技术在硬组织 - 种植体界面上进行组织学和研究。 6(11):4447-4455
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kalve, S., Saini, K., Vissenberg, K., Beeckman, T. and Beemster, G. T. (2015). Transverse Sectioning of Arabidopsis thaliana Leaves Using Resin Embedding . Bio-protocol 5(18): e1592. DOI: 10.21769/BioProtoc.1592.
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