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Immunogold Labeling Analysis of Cell Wall Polysaccharides with Special Reference to (1;3,1;4)-β-D-glucan in Rice Cell Walls
免疫金标记分析水稻细胞壁中的细胞壁多糖尤其是(1;3,1;4)-β-D-葡聚糖   

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参见作者原研究论文

本实验方案简略版
Plant & Cell Physiology
Feb 2015

Abstract

Various types of cell wall compositions have evolved to fulfill a wide range of biological roles during the diversification of land plants. (1;3,1;4)-β-D-glucan (MLG) is a defining feature of the cell walls in the order Poales (Yokoyama and Nishitani, 2004), which has multiple functions associated with metabolic, growth, and defense systems. MLG is also a characteristic component of the matrix polysaccharides that undergo turnover and metabolism, depending on the tissue and the stage of development (Kido et al., 2015). Determining the extracellular localization of MLG is essential for elucidating its functions. Electron microscopy immunogold labeling analysis is a useful technique, which provides an accurate representation of the extracellular distribution of MLG. This strategy is also applicable to various kinds of cell wall polysaccharides, which have key roles in regulating growth and differentiation in each plant species.

Keywords: Electron microscopy (电子显微镜), Cell wall (细胞壁), Immunogold labeling (金标记), Rice (大米), Mixed-linkage glucan (混联葡聚糖)

Materials and Reagents

  1. Gelatin capsule (HF capsule) (Matsuya Co.)
  2. Rice plants (see Note)
  3. Paraformaldehyde (Electron Microscopy Sciences, catalog number: 12300 )
  4. Sodium cacodylate buffer solution (TAAB Laboratories BATCH, catalog number: 90760 )
  5. Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16220 )
  6. Tannic acid (Sigma-Aldrich, catalog number: 403040 )
  7. Phosphate buffer powder (Siyaku, Wako Pure Chemical Industries, catalog number: 167-14491 )
  8. Ethanol (Siyaku, Wako Pure Chemical Industries, catalog number: 057-00456 )
  9. LR White resin (Electron Microscopy Sciences, London Resin Company, catalog number: 14381-CA )
  10. Monoclonal antibody against (1;3,1;4)-β-D-glucan (mouse IgG, Kappa Light) (Biosupplies, catalog number: 400-3 )
    Note: It is named as (1-3;1-4)-β-D-glucan on Biosupplies’ website.
  11. Goat Anti-mouse IgG pAb, Gold 15 nm, EM (BBI Solutions, catalog number: EM.GMHL15 )
  12. Phosphate-buffered salts (Takara Bio Company, catalog number: T900 )
  13. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
  14. Uranyl acetate (Siyaku, Wako Pure Chemical Industries, catalog number: 554-85072 )
  15. Lead stain solution (Sigma-Aldrich, catalog number: 18-0875-2-25 ml-J )
    Note: This reagent may not be available outside Japan, and can be replaced with alternative lead stains. For an alternative, readers are referred to a more general experimental manual for electron microscopy of plant cells (Hall and Hawes, 1991).
  16. Molecular sieves (Sigma-Aldrich, catalog number: M6141 )
  17. Fixative solution (see Recipes)
  18. 2% glutaraldehyde solution (see Recipes)
  19. 100% ethanol solution (see Recipes)
  20. 1x Phosphate-buffered saline (PBS) (see Recipes)

Equipment

  1. Growth chamber (Nippon Medical & Chemical Instruments, model: LH220S )
  2. Ultracut UCT Ultramicrotome (Leica Microsystems)
  3. Diaphragm vacuum pump (Leybold-Heraus, model: Divac 2.2 L )
  4. Desiccator (Sanplatec, model: PC-250K )
  5. Grid mesh (Okenshoji, catalog number: 09-1079 )
  6. Transmission electron microscope (JEOL USA, model: JEM-1400plus )
  7. CCD camera (Olympus soft imaging solutions GmbH, model: Veleta )

Procedure

  1. Cut the rice leaf blade into pieces measuring ≤ 2 mm in length and immediately immerse them in a fixative solution. The fixative solution should be handled under a fume hood.


    Figure 1. Embedding and trimming. a. Equipment and reagent. b. The tissues in the bottom of gelatin capsules filled with fresh 100% LR White resin. c. Polymerization. d. Trimming.

  2. The tissue/fixative is placed in a desiccator, which is connected to a diaphragm vacuum pump. The tissue/fixative is vacuum degasified for 1 min at room temperature using the vacuum pump before releasing the vacuum very slowly.
  3. Incubate the tissue/fixative for 2 h at 4 °C.
  4. Rinse the tissues gently three times with 50 mM sodium cacodylate buffer (pH 7.4) for 15 min each time.
  5. The tissues are dehydrated using a graded ethanol series (70%, 80%, 90%, and three times at 100%) at 4 °C for 30 min in each solution. Swirl occasionally.
  6. Immerse the tissues three times with a 50:50 mixture of 100% (v/v) ethanol and 100% (v/v) LR White resin for 30 min each time at room temperature.
  7. Transfer the tissues to 100% LR White resin and immerse the tissues three times for 1 h each time at room temperature. This step ensures that no residual ethanol will be carried over since other chemical like ethanol or else prevents polymerization of LR resin.
  8. The tissues are settled to the bottom of gelatin capsules filled with fresh 100% LR White resin (Figure 1b). Stand the gelatin capsules in the test-tube rack for polymerization overnight at 50 °C (Figure 1c).
  9. Turn the tissue block upside down and secure it in a UCT ultramicrotome. Chuck and trim the block to expose the tissue surface area up to 1.0 sq mm using a razor blade (Figure 1d).


    Figure 2. Sectioning. a. Sectioning with a ultramicrotome. b. Mounting the ultrathin sections on the grid.

  10. Cut the block to 80 nm with a diamond knife on a UCT ultramicrotome (Figure 2a). Dip up the serial sections floating on water in the boat of the diamond knife with a nickel grid mesh, and mount the ultrathin sections on the grid (Figure 2b).
  11. Incubate the grid with a droplet of the mouse anti-(1;3,1;4)-β-D-glucan antibody (diluted 1:1,000 in a 1% BSA solution, depending on the abundance of the target primary antibody) at room temperature for 90 min (Figure 3a).
  12. Rinse the grid three times with 1% BSA/1x PBS for 1 min each time.
  13. Float the grid on a droplet of goat anti-mouse IgG coupled to 15-nm gold particles 1:20 in 1% BSA/1x PBS at room temperature for 1 h. When detecting another primary antibody, prepare goat anti-mouse IgG coupled to gold particles to match the primary antibody.


    Figure 3. Immunostaining (a) and staining with uranyl acetate (b)

  14. Rinse the grid and the sections three times with 1x PBS.
  15. Immerse the grid and the sections in 2% glutaraldehyde solution and are air-dried under a fume hood.
  16. Immerse the sections in a droplet of 2% uranyl acetate, which had been prepared by diluting with distilled water, for 15 min (Figure 3b).
  17. Rinse the sections with distilled water.
  18. Immerse the sections with Lead stain solution at room temperature for 3 min. Rinse the sections with distilled water.
  19. Observe the sections using a JEOL JEM 1200EX transmission electron microscope at an accelerating voltage of 80 kV.
  20. Digital images (2,048 x 2,048 pixels) are obtained with a CCD camera.

Representative data


Figure 4. Immunogold-based localization of MLG is shown in the leaf blades. a. The images in (a) are of the cell walls of the epidermal cell with a wart-like protuberance (WP). Scale bar = 5 μm. b. A higher magnification of the rectangle in (a). Scale bar = 1 μm. c. The images in (c) are of the cell walls of the cortical sclerenchyma. Scale bar= 5 μm. d. A higher magnification of the rectangle in (c). Scale bar = 1 μm.

Notes

Rice (Oryza sativa L. cv Nipponbare) plants are grown in a growth chamber at 28 °C under a 15/9 h light/dark cycle (light at 150 μmol-2 s-1). This protocol is performed using the fifth leaf blades of the rice plants.

Recipes

  1. Fixative solution
    Prepare fixative solution by dissolving 0.4 g of paraformaldehyde, 0.05 g of tannic acid, and 0.1 ml of 10% glutaraldehyde in 9.9 ml of 50 mM sodium cacodylate buffer (pH 7.4). Heat this solution in the fume hood on the hotplate/stirrer to approx. 70 °C until the solution clears.
  2. 2% glutaraldehyde solution
    Dissolve an appropriate volume of Wako Pure Chemical Industries ready-made powder in distilled water according to manufacturer's protocol, to make a 0.1 M phosphate buffer of pH 7.4 at room temperature. Prepare 2% glutaraldehyde solution by diluting 10% glutaraldehyde in the phosphate buffer.
  3. 100% ethanol solution
    To prepare the 100% ethanol solution, use 100% bulk ethanol with molecular sieves in the bottom of the bottle
  4. 1x PBS
    Dissolve 10 PBS tablets in distilled water to make a total volume of 1,000 ml

Acknowledgments

The electron microscopic analysis was performed by Tokai Electron Microscopy (Nagoya, Japan). This study was supported by the Japan Society for Promotion of Science (JSPS) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan [a Grant-in-Aid for Scientific Research on Innovative Areas “Plant Cell Wall as Information Processing System” (No. 24114001, 24114005) to K. N. and a Grant-in-Aid for Scientific Research (C) (25440124) to R.Y.].

References

  1. Hall, J. L. and Hawes, C. R. (1991). Electron microscopy of plant cells. Academic Press.
  2. Kido, N., Yokoyama, R., Yamamoto, T., Furukawa, J., Iwai, H., Satoh, S. and Nishitani, K. (2015). The matrix polysaccharide (1;3,1;4)-beta-D-glucan is involved in silicon-dependent strengthening of rice cell wall. Plant Cell Physiol 56(2): 268-276.
  3. Yokoyama, R. and Nishitani, K. (2004). Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis. Plant Cell Physiol 45(9): 1111-1121.

简介

各种类型的细胞壁组合物已经发展以在陆地植物的多样化过程中实现广泛的生物学作用。 (1; 3,1; 4)-β-D-葡聚糖(MLG)是Poales(Yokoyama和Nishitani,2004)中的细胞壁的定义特征,其具有与代谢,生长和防御相关的多种功能 系统。 MLG也是经历翻转和代谢的基质多糖的特征组分,这取决于组织和发育阶段(Kido等人,2015)。 确定MLG的细胞外定位对于阐明其功能是必要的。 电子显微镜免疫金标记分析是一种有用的技术,其提供MLG的细胞外分布的精确表示。 该策略也适用于各种细胞壁多糖,其在调节每种植物物种的生长和分化中具有关键作用。

关键字:电子显微镜, 细胞壁, 金标记, 大米, 混联葡聚糖

材料和试剂

  1. 明胶胶囊(HF胶囊)(Matsuya Co.)
  2. 水稻植物(见注)
  3. 多聚甲醛(Electron Microscopy Sciences,目录号:12300)
  4. 二甲胂酸钠缓冲液(TAAB Laboratories BATCH,目录号:90760)
  5. 戊二醛(Electron Microscopy Sciences,目录号:16220)
  6. 鞣酸(Sigma-Aldrich,目录号:403040)
  7. 磷酸盐缓冲液粉末(Siyaku,Wako Pure Chemical Industries,目录号:167-14491)
  8. 乙醇(Siyaku,Wako Pure Chemical Industries,目录号:057-00456)
  9. LR White树脂(Electron Microscopy Sciences,London Resin Company,目录号:14381-CA)
  10. 针对(1; 3,1; 4)-β-D-葡聚糖(小鼠IgG,Kappa Light)(Biosupplies,目录号:400-3)的单克隆抗体
    注意:在Biosupplies的网站上它被命名为(1-3; 1-4)-β-D-葡聚糖。
  11. 山羊抗小鼠IgG pAb,Gold 15nm,EM(BBI Solutions,目录号:EM.GMHL15)
  12. 磷酸盐缓冲盐(Takara Bio Company,目录号:T900)
  13. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2058)
  14. 乙酸乙酯(Siyaku,Wako Pure Chemical Industries,目录号:554-85072)
  15. 铅染色溶液(Sigma-Aldrich,目录号:18-0875-2-25ml-J) 注意:此试剂可能不在日本以外提供,可以用替代铅污染物替代。作为替代,读者参考用于植物细胞的电子显微镜的更一般的实验手册(Hall和Hawes,1991)。
  16. 分子筛(Sigma-Aldrich,目录号:M6141)
  17. 固定解决方案(参见配方)
  18. 2%戊二醛溶液(见配方)
  19. 100%乙醇溶液(见配方)
  20. 1x磷酸盐缓冲盐水(PBS)(参见配方)

设备

  1. 生长室(Nippon Medical& Chemical Instruments,型号:LH220S)
  2. Ultracut UCT超薄切片机(Leica Microsystems)
  3. 隔膜真空泵(Leybold-Heraus,型号:Divac 2.2 L)
  4. 干燥器(Sanplatec,型号:PC-250K)
  5. 网格(Okenshoji,目录号:09-1079)
  6. 透射电子显微镜(JEOL USA,型号:JEM-1400plus)
  7. CCD照相机(Olympus软成像解决方案有限公司,型号:Veleta)

程序

  1. 将水稻叶片切成长度≤2mm的块,并立即将其浸入固定溶液中。固定溶液应在通风橱下处理。


    图1.嵌入和修剪。 a。设备和试剂。 b。用新鲜的100%LR白色树脂填充明胶胶囊底部的组织。 C。聚合。 d。修剪。

  2. 将组织/固定剂置于干燥器中,其连接至隔膜真空泵。在非常缓慢地释放真空之前,使用真空泵在室温下将组织/固定剂真空脱气1分钟。
  3. 在4℃下孵育组织/固定剂2小时
  4. 用50mM二甲胂酸钠缓冲液(pH 7.4)轻轻冲洗组织三次,每次15分钟。
  5. 使用梯度乙醇系列(70%,80%,90%,并且在100%下三次)在4℃下将组织脱水30分钟。偶尔旋转。
  6. 用100%(v/v)乙醇和100%(v/v)LR White树脂的50:50混合物将组织浸泡三次,每次30分钟。
  7. 转移组织到100%LR白色树脂和浸泡组织三次,每次1小时,在室温下。该步骤确保不会残留残留的乙醇,因为其它化学品如乙醇或者防止LR树脂的聚合
  8. 将组织沉降到填充有新鲜的100%LR白色树脂的明胶胶囊的底部(图1b)。将试管架中的明胶胶囊置于50℃聚合过夜(图1c)。
  9. 将组织块倒置,并将其固定在UCT超薄切片机。使用剃刀刀片(图1d)夹紧并修剪该块以暴露组织表面积达1.0平方毫米。


    图2.剖切。用超薄切片机切片。 b。将超薄部分安装在网格上。

  10. 用金刚石刀在UCT超薄切片机上将块切成80nm(图2a)。使用镍网格网将浮动在金刚石刀船中的水上的串联部分浸没,并将超薄部分安装在网格上(图2b)。
  11. 用一滴小鼠抗(1; 3,1; 4)-β-D-葡聚糖抗体(在1%BSA溶液中稀释1:1,000,取决于靶一级抗体的丰度)孵育网格,室温90分钟(图3a)。
  12. 用1%BSA/1×PBS冲洗网格三次,每次1分钟
  13. 将网格浮在与1-BSA/1×PBS中的15-nm金颗粒1:20偶联的山羊抗小鼠IgG的小滴上,在室温下1小时。当检测其他一抗时,制备与金颗粒偶联的山羊抗小鼠IgG以匹配一抗

    图3.免疫染色(a)和用乙酸铀酰染色(b)

  14. 用1x PBS冲洗网格和切片三次。
  15. 将网格和切片浸入2%戊二醛溶液中,并在通风橱中风干
  16. 将切片浸入用蒸馏水稀释的2%乙酸双氧铀液滴中15分钟(图3b)。
  17. 用蒸馏水冲洗切片。
  18. 浸渍切片与铅染色溶液在室温下3分钟。用蒸馏水冲洗切片。
  19. 使用JEOL JEM 1200EX透射电子显微镜在80kV的加速电压下观察切片。
  20. 用CCD照相机获得数字图像(2048×2048像素)。

代表数据


图4.在叶片中显示了MLG的基于免疫金的定位。。 (a)中的图像是具有疣状突起(WP)的表皮细胞的细胞壁。比例尺=5μm。 b。 (a)中矩形的放大率更高。比例尺=1μm。 C。 (c)中的图像是皮层厚壁的细胞壁的图像。比例尺=5μm。 d。 (c)中矩形的放大率更高。比例尺=1μm。

笔记

在28℃,在15/9h光/暗循环(150μmol-2μm)下,在生长室中生长水稻(水稻,日本晴> s -1 )。该方案使用水稻植物的第五叶叶进行。

食谱

  1. 固定解决方案
    通过将0.4g多聚甲醛,0.05g丹宁酸和0.1ml 10%戊二醛溶解在9.9ml 50mM二甲胂酸钠缓冲液(pH 7.4)中制备固定溶液。将该溶液在加热板/搅拌器上的通风橱中加热至约30℃。 70°C,直到溶液澄清
  2. 2%戊二醛溶液
    根据制造商的方案,将适当体积的Wako Pure Chemical Industries现成的粉末溶解在蒸馏水中,在室温下制备pH 7.4的0.1M磷酸盐缓冲液。通过稀释磷酸盐缓冲液中的10%戊二醛制备2%戊二醛溶液。
  3. 100%乙醇溶液
    为了制备100%乙醇溶液,在瓶子底部使用100%含有分子筛的大量乙醇
  4. 1x PBS
    将10个PBS片溶于蒸馏水中,使总体积为1000ml

致谢

电子显微镜分析由Tokai Electron Microscopy(Nagoya,Japan)进行。本研究由日本科学促进会(JSPS)和教育,文化,体育科学技术部(MEXT),日本[创新地区科学研究资助"植物细胞壁作为信息处理系统"(No.24114001,24114005)和KY的科学研究授权(C)(25440124)。

参考文献

  1. Hall,J.L。和Hawes,C.R。(1991)。植物细胞的电子显微镜。 学术出版社。
  2. Kido,N.,Yokoyama,R.,Yamamoto,T.,Furukawa,J.,Iwai,H.,Satoh,S.and Nishitani,K。 基质多糖(1; 3,1; 4)-β-D-葡聚糖参与硅依赖性强化稻细胞壁。植物细胞生理学56(2):268-276。
  3. Yokoyama,R。和Nishitani,K。(2004)。 植物细胞壁多样性的基因组基础。水稻和拟南芥中基因家族的比较方法植物细胞生理学 45(9):1111-1121。
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引用:Yokoyama, R., Kido, N., Yamamoto, T., Furukawa, J., Iwai, H., Satoh, S. and Nishitani, K. (2016). Immunogold Labeling Analysis of Cell Wall Polysaccharides with Special Reference to (1;3,1;4)-β-D-glucan in Rice Cell Walls. Bio-protocol 6(5): e1748. DOI: 10.21769/BioProtoc.1748.
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