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Plastic Embedding of Arabidopsis Stem Sections
拟南芥干细胞切片的树脂包埋

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Abstract

The inflorescence stem of the flowering plant Arabidopsis thaliana (thale cress) is an excellent model system to investigate plant vascular tissue patterning and development. Plant vasculature is a complex conducting tissue arranged in strands called vascular bundles, formed by xylem (tissue that carries water) and phloem (tissue that carries photosynthates and signaling molecules). Xylem and phloem are originated from cell division of the meristematic cells of the vascular cambium. In Arabidopsis the flowering stem elongates about three weeks after germination. At this stage it is possible to visualize defects in its development and morphology. Here we describe a protocol to embed in plastic (resin) stem segments either freshly dissected from living plants or previously assayed for β-glucuronidase. This protocol provides an excellent cellular morphology ideal to visualize stem cell types including those of vascular bundles using high-resolution light microscopy.

Keywords: Arabidopsis(拟南芥), Resin sections(树脂切片), Stem(茎), Vascular tissue phenotype(血管组织表型), Plastic sections(塑料型材)

Materials and Reagents

  1. 30 day-old Arabidopsis stem fragments
  2. 90% acetone
  3. 4% glutaraldehyde
  4. 0.1% Tween 20
  5. Absolute ethanol
  6. Historesin standard kit (Reichert-Jung, catalog number: 7O-2218-500)
    Components:
    1. Basic resin liquid (Figure 2A)
    2. Activator powder
    3. Hardener
  7. Cresyl violet acetate (Sigma-Aldrich, catalog number: C-5042)
  8. Preinfiltration solution (see Recipes)
  9. Infiltration solution (see Recipes)
  10. Embedding medium (see Recipes)
  11. Cresyl violet staining medium (see Recipes)

Equipment

  1. Vacuum pump (Figure 1)
  2. Vacuum jar (Figure 1)
  3. Histomold trays (6 x 8 mm) (Leica Microsystems, catalog number: 14702218311)
  4. Histomold trays (13 x 19 mm) (Leica Microsystems, catalog number: 14702218313)
  5. Specimen Holder adapter (Leica Microsystems, catalog number: 14702218310)
  6. Drying oven DO-90 (Raypa)
  7. Motorized microtome Leica RM2155 (Leica Microsystems)
  8. Water/ethanol bath HI 1210 (Leica Microsystems)
  9. Tungsten carbide disposable blades TC-65 (Leica Microsystems, catalog number: 140216266379)
  10. Glass slides Superfrost ultra plus (Menzel-Glasër, catalog number: J3800AMNZ)
  11. Surgipath micromount (Leica Microsystems, catalog number: 3801731)
  12. Coverslip (24 x 60) (Menzel-Glasër, catalog number: BB024060A1)


    Figure 1. Vacuum jar and vacuum pump used for tissue infiltration

Procedure

  1. Collect stem fragments (5 to 10 mm) of 30 day-old Arabidopsis plants of the main inflorescence from immediately above the uppermost rosette leaf in a 15 ml falcon (Figure 2A).
  2. Incubate for 30 min in 90% acetone.
  3. Wash twice with water by emptying the recipient and filling it with fresh water.
  4. Cover with 4% glutaraldehyde, 0.1% Tween 20 and vacuum infiltrate for 15 min.
  5. Leave at 4 °C overnight or longer. Stems can be stored in this fixative.
  6. Wash twice with water.
  7. Dehydrate in ethanol series up to 100% ethanol:
    30% ethanol (3 h)
    50% ethanol (3 h)
    75% ethanol (3 h)
    95% ethanol (3 h)
    100% ethanol (overnight)
  8. Incubate the stems in Preinfiltration solution overnight (minimum) at 4 °C.
  9. Incubate the stems in Infiltration solution at 4 °C (overnight minimum, up to two weeks).
  10. Fill the bottom of HISTORESIN MOLD TRAYS with embedding medium (Figures 2B, 2C).
  11. Immerse and orient the specimens in the mold trays (Figures 2D, 2E).
    Put the block holder adapter on the mold before polymerization so that it forms a single piece together with the block (Figures 2F, 2G).
  12. Allow to polymerize overnight at room temperature.
  13. Take out the block plus adaptor from the mold (Figures 2H-J).


    Figure 2. Making a resin block. A. Reactives needed for block making. B, C. The mold trays are filled with embedding medium. D, E. The specimens are immersed and orient in the mold trays. F, G. The block holder adapter is set on the mold before polymerization. H-J. The block plus adaptor is taken out of the mold.

  14. Fix it to the mobile head of the microtome (Figure 3A-3C).
  15. Cut 3-8 µm sections of resin-embedded specimens using a motorized microtome Leica RM2155 with tungsten carbide blades TC-65 (Figures 3C, 3D, Video 1).

    Video 1. Cutting and slides

    To play the video, you need to install a newer version of Adobe Flash Player.

    Get Adobe Flash Player


  16. Float sections in a 50 °C water/ethanol bath for a few seconds until the section flattens (Figures 3E, 3F, Video 1). About 40 ml of 100% ethanol per liter of water are added to restrict the rapid movements of the historesin sections occurring when floating in water.
  17. Collect sections on a glass slide (Figure 2G, Video 1) and allow to dry in an oven overnight at 50 °C.
  18. Optional staining step (only when a contrast staining solution is needed).
  19. Immerse the slides in cresyl violet staining medium 2-5 min.
  20. Wash with distilled water 2 min.
  21. Dry the excess of moisture from the slides using a kimwipe.
  22. Dry in oven for 15 min at 50 °C.
  23. Mount sections in Surgipath micromount.
  24. Cover with a coverslip.


    Figure 3. Sectioning a resin block. A-C. The block is fixed to the mobile head of the microtome. D. 3-8 µm sections of resin-embedded specimens are cut using a motorized microtome. E. Sections are collected with tweezers. F. Sections are put to float in a 50 °C water bath for a few seconds until the section flattens. G. Sections are collected on a glass slide.


    Figure 4. Arabidopsis stem section stained with cresyl violet, prepared as described in this protocol

Notes

  1. To obtain transversal stem sections a 2-step process is used. First, the stems are horizontally laid down in the mold, at the bottom of the surface, and a first block is made. Then this block is trimmed to leave only a piece of resin containing the stem. This block is oriented vertically in a second mold, which is used to produce a second block where the stem will be properly oriented for sectioning.
  2. This procedure can be applied to other samples, both from animal and plant origins. Generally, plant tissues require longer infiltration times (minimum 2-3 days), as compared to animal tissues (where normally an overnight infiltration time is sufficient).

Recipes

  1. Preinfiltration solution
    50% absolute ethanol
    50% infiltration solution
  2. Infiltration solution
    50 ml basic resin/liquid
    1 packet (0.5 g) activator
    Stir with a magnetic stirrer until completely dissolved
    It can be stored at 4 °C for several weeks.
  3. Embedding medium
    15 ml infiltration solution
    1 ml hardener
    Freshly made
    Mix and use immediately
  4. Cresyl violet staining medium
    1% cresyl violet acetate in distilled water
    100 mg of cresyl ciolet acetate
    10 ml of distilled water

Acknowledgments

This work was supported by the Ministerio de Ciencia y Tecnología (BIO2011-25687). This protocol was adapted from Giménez et al. (2001) and Chevalier et al. (2014). We thank Inés Poveda for photographic work.

References

  1. Chevalier, F., Nieminen, K., Sanchez-Ferrero, J. C., Rodriguez, M. L., Chagoyen, M., Hardtke, C. S. and Cubas, P. (2014). Strigolactone promotes degradation of DWARF14, an alpha/beta hydrolase essential for strigolactone signaling in Arabidopsis. Plant Cell 26(3): 1134-1150.
  2. Gimenez, E., Giraldo, P., Jeffery, G. and Montoliu, L. (2001). Variegated expression and delayed retinal pigmentation during development in transgenic mice with a deletion in the locus control region of the tyrosinase gene. Genesis 30(1): 21-25.

简介

开花植物的开花茎(Arabidopsis thaliana)(thale cress)是研究植物血管组织成像和发育的优良模型系统。 植物脉管系统是一种复杂的导电组织,其排列成由木质部(携带水的组织)和韧皮部(携带光合产物和信号分子的组织)形成的称为维管束的线束。 木质部和韧皮部起源于血管形成层的分生细胞的细胞分裂。 在拟南芥中,开花茎在萌发后约三周延长。 在这个阶段,可以看到缺陷的发展和形态。 在这里我们描述嵌入塑料(树脂)茎段的新方案从活植物或以前测定β-葡糖醛酸糖苷酶的协议。 这个协议提供了一个优秀的细胞形态理想的可视化干细胞类型,包括那些维管束使用高分辨率光学显微镜。

关键字:拟南芥, 树脂切片, 茎, 血管组织表型, 塑料型材

材料和试剂

  1. 30天的拟南芥茎片段
  2. 90%丙酮
  3. 4%戊二醛
  4. 0.1%Tween 20
  5. 绝对乙醇
  6. 组织蛋白酶标准试剂盒(Reichert-Jung,目录号:70-2218-500)
    组件:
    1. 碱性树脂液(图2A)
    2. 活化剂粉末
    3. 固化剂
  7. 甲酚紫醋酸酯(Sigma-Aldrich,目录号:C-5042)
  8. 预过滤溶液(参见配方)
  9. 渗透解决方案(参见配方)
  10. 嵌入介质(参见配方)
  11. 甲酚紫染色介质(参见配方)

设备

  1. 真空泵(图1)
  2. 真空罐(图1)
  3. Histomold托盘(6×8mm)(Leica Microsystems,目录号:14702218311)
  4. Histomold托盘(13×19mm)(Leica Microsystems,目录号:14702218313)
  5. 样品夹适配器(Leica Microsystems,目录号:14702218310)
  6. 干燥炉DO-90(Raypa)
  7. 电动切片机Leica RM2155(Leica Microsystems)
  8. 水/乙醇浴HI 1210(Leica Microsystems)
  9. 碳化钨一次性刀片TC-65(Leica Microsystems,目录号:140216266379)
  10. 玻璃幻灯片Superfrost ultra plus(Menzel-Glasër,目录号:J3800AMNZ)
  11. Surgipath微型装载(Leica Microsystems,目录号:3801731)
  12. 盖玻片(24 x 60)(Menzel-Glasër,目录号:BB024060A1)


    图1.用于组织浸润的真空瓶和真空泵

程序

  1. 从15毫升猎鹰(图2A)中最上面的莲座叶上方收集主花序的30天的拟南芥植物的茎片段(5至10mm)。
  2. 在90%丙酮中孵育30分钟
  3. 用水清洗两次,清空收件人并用新鲜水填充
  4. 用4%戊二醛,0.1%吐温20覆盖,并真空浸润15分钟
  5. 在4℃下放置过夜或更长时间。 茎可以存储在此固定剂中。
  6. 用水洗涤两次。
  7. 在乙醇系列中至多100%乙醇脱水:
    30%乙醇(3小时) 50%乙醇(3小时) 75%乙醇(3小时) 95%乙醇(3小时) 100%乙醇(过夜)
  8. 在4°C孵育过夜(最小)预浸液中的茎。
  9. 在4℃下在浸润溶液中孵育茎(最小过夜,长达两周)。
  10. 填充HISTORESIN模具托盘的底部与包埋介质(图2B,2C)。
  11. 将样品浸入和定向在模具托盘中(图2D,2E)。
    在聚合之前将块支架适配器放在模具上,使其与块一起形成单件(图2F,2G)。
  12. 允许在室温下聚合过夜。
  13. 从模具中取出块加适配器(图2H-J)。

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    图2.制作树脂块。 A.块制作所需的反应物。 B,C。模具托盘填充有包埋介质。 D,E。将样品浸入并在模具托盘中定向。 F,G。在聚合之前将块支架适配器设置在模具上。 H-J。 块加适配器从模具中取出。

  14. 将其固定到切片机的移动头(图3A-3C)。
  15. 使用具有碳化钨刀片TC-65的机动切片机Leica RM2155切割树脂嵌入样本的3-8μm切片(图3C,3D,视频1)。

    视频1.切割和幻灯片
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    获取Adobe Flash Player

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    获取Adobe Flash Player

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  16. 在50℃水/乙醇浴中漂浮切片几秒钟,直到切片变平(图3E,3F,视频1)。 加入约40ml的100%乙醇/升水,以限制漂浮在水中时发生的组织蛋白酶切片的快速移动。
  17. 收集玻片上的切片(图2G,视频1),并允许在烘箱中在50℃下干燥过夜。
  18. 可选染色步骤(仅当需要造影染色溶液时)
  19. 将载玻片浸入甲酚紫染色培养基2-5分钟
  20. 用蒸馏水清洗2分钟。
  21. 使用kimwipe从幻灯片中干燥过量的水分。
  22. 在50℃下在烘箱中干燥15分钟
  23. 在Surgipath微型安装中安装部分
  24. 用盖玻片盖。


    图3.切割树脂块。 A-C。 块体固定到切片机的移动头部。 D.使用机动切片机切割3-8μm切片的树脂包埋的样品。 E.用镊子收集切片。 F.将切片在50℃水浴中漂浮几秒钟直到切片变平。 G.在玻片上收集切片。


    图4. 拟南芥 茎部分用甲酚紫染色,按本协议所述制备

笔记

  1. 为了获得横向茎段,使用2步法。 首先,杆水平地放置在模具中,在表面的底部,并且制造第一块。 然后修剪该块以仅留下含有茎的一片树脂。 该块在第二模具中垂直定向,其用于产生第二块,其中杆将被适当地定向以用于切片。
  2. 该程序可以应用于来自动物和植物来源的其它样品。 通常,与动物组织(其中通常过夜渗透时间足够)相比,植物组织需要更长的浸润时间(最少2-3天)。

食谱

  1. 预先过滤溶液
    50%无水乙醇 50%浸润溶液
  2. 浸润溶液
    50ml碱性树脂/液体
    1包(0.5克)活化剂
    用磁力搅拌器搅拌直至完全溶解
    它可以在4℃下储存数周。
  3. 嵌入媒介
    15ml浸润溶液
    1 ml硬化剂
    新鲜制作
    立即混合并使用
  4. 甲酚紫染色介质
    1%甲酚紫乙酸酯在蒸馏水中的溶液 100mg甲酚红醋酸盐 10ml蒸馏水

致谢

这项工作得到了西班牙部长会议(BIO2011-25687)的支持。 该方案改编自Giménez等人(2001)和Chevalier等人(2014)。 我们感谢InésPoveda的摄影作品。

参考文献

  1. Chevalier,F.,Nieminen,K.,Sanchez-Ferrero,J.C.,Rodriguez,M.L.,Chagoyen,M.,Hardtke,C.S.and Cubas,P.(2014)。 Strigolactone促进 降解DWARF14,一种拟南芥中独脚金内酯信号传导所必需的α/β水解酶。 植物细胞26(3):1134-1150。
  2. Gimenez,E.,Giraldo,P.,Jeffery,G.and Montoliu,L。(2001)。 斑驳表达和延迟的视网膜色素沉着在转基因小鼠的发育过程中,在基因座控制区的 酪氨酸酶基因。 30(1):21-25。
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引用:Chevalier, F., Iglesias, S. M., Sánchez, Ó. J., Montoliu, L. and Cubas, P. (2014). Plastic Embedding of Arabidopsis Stem Sections. Bio-protocol 4(20): e1261. DOI: 10.21769/BioProtoc.1261.
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