Cytohistochemical Determination of Calcium Deposition in Plant Cells
植物细胞中钙沉积的组织化学测定
发布: 2016年01月20日第6卷第2期 DOI: 10.21769/BioProtoc.1709 浏览次数: 8004
评审: Marisa RosaRumen IvanovAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
May 2015
Abstract
Calcium plays important roles in maintaining plant cellular structure and also acts as a key secondary messenger in intercellular signaling. Thirty years ago, methods of detecting calcium in sub-cellular level had been established (Stockwell and Hanchey, 1982; Borgers et al., 1982) and reviewed extensively (Wick and Heplerm, 1982). We had used the method of testing calcium localization in salt tolerance improved transgenic alfalfa plant (Zhang and Wang, 2015). Here, we describe the protocol of testing calcium deposition by staining with potassium pyroantimonate (PPA) in detail, which was adapted from former reports (Stockwell and Hanchey, 1982; Borgers et al., 1982). The principle of this protocol is that the Ca2+ can react with antimonite and from black granules, which can be observed under a transmission electron microscope. The protocol includes common micromanipulation techniques of plant tissue, observation with a transmission electron microscope and photography.
Keywords: Calcium (钙)
Localization (定位)
Antimonite (辉锑矿)
Materials and Reagents
- Grids (Sigma-Aldrich, catalog number: G5526 ) for transmission electron microscopy
- 200 μl centrifuge tubes (Eppendorf or other brand)
- 1.5 ml centrifuge tubes (Eppendorf or other brand)
- 50 ml centrifuge tubes (Eppendorf or other brand)
- 0.22 μm Millipore filter unit (Merck Millipore Corporation)
- Plant root tips (cut into ~2 mm in size)
- 25% Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
- Disodium hydrogen phosphate dodecahydrate (Na2HPO4) (Sinopharm Chemical Reagent Co., catalog number: 10039-32-4 )
- Potassium pyroantimonate (PPA) (Sigma-Aldrich, catalog number: 60500 )
- 1 M NaOH (Sinopharm Chemical Reagent Co., catalog number: 10019718 )
- Acetone (100%) (Sigma-Aldrich, catalog number: 69508 )
- Ethanol (100%) (Sinopharm Chemical Reagent Co., catalog number: 100092690 )
- Ethylene glycol-O, O′-bis (2-aminoethyl)-1 N, N, N′, N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
- 1 M HCl (Sinopharm Chemical Reagent Co., catalog number: 10011061 )
- Epon 812 (Sigma-Aldrich, catalog number: 45346 )
- Formvar solution (Sigma-Aldrich, catalog number: 09823 )
Equipment
- Refrigerator (4 °C)
- Incubator
- Vacuum equipment (Vacuum pump connect to a desiccator)
- pH meter
- Transmission electron microscope (Hitachi High-Technologies Europe GmbH, model: H-7500 )
- Ultramicrotome (Leica Microsystems, model: EM UC6 )
- Tweezers (sharp tip)
- Glass beakers
- Glass stirring rods
Procedure
文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Zhang, W. and Wang, T. (2016). Cytohistochemical Determination of Calcium Deposition in Plant Cells. Bio-protocol 6(2): e1709. DOI: 10.21769/BioProtoc.1709.
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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 > 植物生理学 > 离子分析
生物化学 > 其它化合物 > 离子 > 钙
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