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
发布: 2016年03月05日第6卷第5期 DOI: 10.21769/BioProtoc.1748 浏览次数: 9621
评审: Tie LiuCarsten AdeValérie Cornuault
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参见作者原研究论文
The authors used this protocol in:
Feb 2015
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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
- Gelatin capsule (HF capsule) (Matsuya Co.)
- Rice plants (see Note)
- Paraformaldehyde (Electron Microscopy Sciences, catalog number: 12300 )
- Sodium cacodylate buffer solution (TAAB Laboratories BATCH, catalog number: 90760 )
- Glutaraldehyde (Electron Microscopy Sciences, catalog number: 16220 )
- Tannic acid (Sigma-Aldrich, catalog number: 403040 )
- Phosphate buffer powder (Siyaku, Wako Pure Chemical Industries, catalog number: 167-14491 )
- Ethanol (Siyaku, Wako Pure Chemical Industries, catalog number: 057-00456 )
- LR White resin (Electron Microscopy Sciences, London Resin Company, catalog number: 14381-CA )
- 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. - Goat Anti-mouse IgG pAb, Gold 15 nm, EM (BBI Solutions, catalog number: EM.GMHL15 )
- Phosphate-buffered salts (Takara Bio Company, catalog number: T900 )
- Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2058 )
- Uranyl acetate (Siyaku, Wako Pure Chemical Industries, catalog number: 554-85072 )
- 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). - Molecular sieves (Sigma-Aldrich, catalog number: M6141 )
- Fixative solution (see Recipes)
- 2% glutaraldehyde solution (see Recipes)
- 100% ethanol solution (see Recipes)
- 1x Phosphate-buffered saline (PBS) (see Recipes)
Equipment
- Growth chamber (Nippon Medical & Chemical Instruments, model: LH220S )
- Ultracut UCT Ultramicrotome (Leica Microsystems)
- Diaphragm vacuum pump (Leybold-Heraus, model: Divac 2.2 L )
- Desiccator (Sanplatec, model: PC-250K )
- Grid mesh (Okenshoji, catalog number: 09-1079 )
- Transmission electron microscope (JEOL USA, model: JEM-1400plus )
- CCD camera (Olympus soft imaging solutions GmbH, model: Veleta )
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
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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分类
植物科学 > 植物生物化学 > 糖类
生物化学 > 糖类 > 多糖
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