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Cytohistochemical Determination of Calcium Deposition in Plant Cells
植物细胞中钙沉积的组织化学测定   

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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 electronic microscope. The protocol includes common micromanipulation techniques of plant tissue, observation with a transmission electronic microscope and photography.

Keywords: Calcium(钙), Localization(定位), Antimonite(辉锑矿)

Materials and Reagents

  1. Grids (Sigma-Aldrich, catalog number: G5526 ) for transmission electron microscopy
  2. 200 μl centrifuge tubes (Eppendorf or other brand)
  3. 1.5 ml centrifuge tubes (Eppendorf or other brand)
  4. 50 ml centrifuge tubes (Eppendorf or other brand)
  5. 0.22 μm Millipore filter unit (Merck Millipore Corporation)
  6. Plant root tips (cut into ~2 mm in size)
  7. 25% Glutaraldehyde (Sigma-Aldrich, catalog number: G6257 )
  8. Disodium hydrogen phosphate dodecahydrate (Na2HPO4) (Sinopharm Chemical Reagent Co., catalog number: 10039-32-4 )
  9. Potassium pyroantimonate (PPA) (Sigma-Aldrich, catalog number: 60500 )
  10. 1 M NaOH (Sinopharm Chemical Reagent Co., catalog number: 10019718 )
  11. Acetone (100%) (Sigma-Aldrich, catalog number: 69508 )
  12. Ethanol (100%) (Sinopharm Chemical Reagent Co., catalog number: 100092690 )
  13. Ethylene glycol-O, O′-bis (2-aminoethyl)-1 N, N, N′, N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E3889 )
  14. 1 M HCl (Sinopharm Chemical Reagent Co., catalog number: 10011061 )
  15. Epon 812 (Sigma-Aldrich, catalog number: 45346 )
  16. Formvar solution (Sigma-Aldrich, catalog number: 09823 )

Equipment

  1. Refrigerator (4 °C)
  2. Incubator
  3. Vacuum equipment (Vacuum pump connect to a desiccator)
  4. pH meter
  5. Transmission electronic microscope (Hitachi High-Technologies Europe GmbH, model: H-7500 )
  6. Ultramicrotome (Leica Microsystems, model: EM UC6 )
  7. Tweezers (sharp tip)
  8. Glass beakers
  9. Glass stirring rods

Procedure

  1. Preparation of PPA staining solution
    1. Making 4% PPA solution: Dissolve 8.0 g potassium pyroantimonate (PPA) in the 400 mM Na2HPO4 (pH 7.6) by heating to 90-95 °C and with continuous agitation. Then after, filter the solution through a 0.22 μm Millipore filter, then keep it in 4 °C refrigerator before use (see Notes 1).
    2. Making PPA staining fluid (50 ml): Mix 25 ml 4% PPA solution with 6 ml 25% Glutaraldehyde, then fill up with 19 ml of sterile H2O (see Notes 2).
  2. Fixation and rinsing: Collect plant root tips that grown on 1/2 MS medium and rinse with distilled water several times, then cut rip tips with scalpel in size of about 2 mm and quickly put it in cold PPA staining fluid in a 1.5 ml centrifuge tube. If the plant materials are floating, give a vacuum treatment (0.8 Mpa, 10 min) till the materials sink to the bottom of tube, fixation at 4 °C, overnight. Discard PPA staining fluid, by rinsing with 1% PPA solution for 3 times, each time for 5 min.
  3. Dehydration: Then after, dehydrate the fixed material in a graded cold acetone serials (30%, 50%, 70%, 90%, 100%, 100%), and 15 min each time.
  4. Penetration: After dehydration, treat the materials with a serial mixtures of Epon 812: pure acetone (1:3, 1:1 and 3:1) for 12 h each time; and pure Epon 812 mixture (35-40 °C) of 12 h.
  5. Embed: Add fresh Epon 812 mixture to clean 200 μl eppendorf tube, transfer root tip carefully and organize the root tip and let it merged in the embedding mixture but not touch to wall of tube with a needle (Figure 1A). Then put the tubes in an incubator, 35 °C for 12 h, then 45 °C for 12 h and 60 °C for 24 h, till the resin is well solidified. Cool down at room temperature for about 1 h.
  6. Coat nickel grids: Coat nickel grids with formvar can be done by merging the grids in formvar solution for several second then dry on filter paper for about 1 to 2 h; or put a drop of formvar solution directly on the nickel grids on a clean grass slide then let it dry down naturally, it takes about 2 h.
  7. Sectioning: After trimming the embedded block with a sharp knife (or a block trimmer) properly, cut the block with an ultra-cut microtome to generate 100 nm thick ultrathin sections and mounted onto 200-mesh formvar-coated nickel grids (Figure 1B). Dry under a lamp for about 1 h.
  8. Microscopy: Check the samples with a transmission electron microscope. Calcium deposition shows black granules or aggregates (Figure 1C-D; Notes 3).


    Figure 1. Representative pattern of sampling and calcium deposition in the root-tip cells of alfalfa. A. Illustration of sample in embedding mixture; B. Illustration of sample section on nickel grid; C. Cortical cell walls of rstB transgenic alfalfa showed large aggregates were deposited on remnants of membranes and cytoplasm. D. The epidermal cell walls of wt plant showed weak calcium staining. The deposited granules are smaller. Calcium deposition was indicated with solid red arrows. Lipid bodies (droplets) were indicated with blue dotted arrows. The scale bars represent 2 μm.

Notes

  1. For plant cells fixation the concentration of Glutaraldehye could range from 2.5% - 4%. In this protocol 3.0% Glutaraldehye was used.
  2. For calcium staining, PPA concentration could range from 0.5% to 2.5% in different protocols.
  3. To confirm the observed granules are calcium deposits, the section could be treated with 0.1 M EGTA (pH 8.0) at 60 °C for 30 min, and check again. Calcium granules could be removed by EGTA treatment (Stockwell and Hanchey, 1982).

Recipes

  1. 1 M Na2HPO4 (pH 7.6)
    Dissolve 138 g NaH2PO4.H2O in 1 L water and dissolve 142 g of Na2HPO4 in 1 L water to make their 1 M stock solution
    During the time, test and modify the pH value with NaOH or HCl to 7.6
  2. 400 mM Na2HPO4 (pH 7.6)
    Mix 338 ml of 1 M Na2HPO4 and 62 ml of 1 M NaH2PO4 and add water to final 1 L
  3. 200 mM Na2HPO4 (pH 7.6)
    Dilute 400 mM Na2HPO4 with equal volume distilled water
  4. 4% PPA solution
    In fuming-hood, dissolve 8.0 g potassium pyroantimonate (PPA) in the 400 mM Na2HPO4 (pH 7.6) by heating to 90-95 °C and continuous agitation.
  5. PPA staining fluid (50 ml)
    Mix 4% PPA solution 25 ml, 25% Glutaraldehyde 6 ml and 19 ml sterile H2O
  6. 0.1 M EGTA
    Add 18.61 g EGTA, 2.0 g NaOH in about 80 ml water with continue stirring till dissolved completely
    Add water to 100 ml
    Modify pH value to 8.0
  7. 1 M NaOH
    Dissolve 4.0 g NaOH in 100 ml water
  8. 1 M HCl
    Take 8.36 ml 37% HCl and add water to 100 ml

Acknowledgments

This protocol was an adaption from Stockwell and Hanchey (1982) and Borgers et al. (1982). The work was supported by National Natural Science Foundation of China (31472140). We are grateful to Mrs. H. Hao for her skillful technical assistance in calcium localization tests.

References

  1. Borgers, M., Thone, F., Verheyen, A. and Ter Keurs, H. E. (1984). Localization of calcium in skeletal and cardiac muscle. Histochem J 16(3): 295-309.
  2. Stockwell, V. and Hanchey, P. (1982). Cytohistochemical techniques for calcium localization and their application to diseased plants. Plant Physiol 70(1): 244-251.
  3. Wick, S. M. and Hepler, P. K. (1982). Selective localization of intracellular Ca2+ with potassium antimonate. J Histochem Cytochem 30(11): 1190-1204.
  4. Zhang, W. J. and Wang, T. (2015). Enhanced salt tolerance of alfalfa (Medicago sativa) by rstB gene transformation. Plant Sci 234: 110-118.

简介

钙在维持植物细胞结构中起重要作用,并且还在细胞间信号传导中作为关键的第二信使。三十年前,已经建立了在亚细胞水平检测钙的方法(Stockwell和Hanchey,1982; Borgers等人,1982)并广泛地综述(Wick和Heplerm,1982)。我们使用测试钙定位的方法在耐盐改良的转基因苜蓿植物中(Zhang和Wang,2015)。在这里,我们描述了通过用详细的焦锑酸钾(PPA)染色测试钙沉积的方案,其改编自以前的报道(Stockwell和Hanchey,1982; Borgers等人,1982)。该方案的原理是Ca 2+ 2+可以与锑矿和黑色颗粒反应,这可以在透射电子显微镜下观察到。该协议包括植物组织的常见显微操作技术,用透射电子显微镜和照相观察。

关键字:钙, 定位, 辉锑矿

材料和试剂

  1. 用于透射电子显微镜的网格(Sigma-Aldrich,目录号:G5526)
  2. 200μl离心管(Eppendorf或其他品牌)
  3. 1.5 ml离心管(Eppendorf或其他品牌)
  4. 50 ml离心管(Eppendorf或其他品牌)
  5. 0.22μm微孔过滤器单元(Merck Millipore Corporation)
  6. 植物根尖(切成?2 mm大小)
  7. 25%戊二醛(Sigma-Aldrich,目录号:G6257)
  8. 磷酸氢二钠十二水合物(Na 2 HPO 4)(Sinopharm Chemical Reagent Co.,目录号:10039-32-4)
  9. 高锑酸钾(PPA)(Sigma-Aldrich,目录号:60500)
  10. 1M NaOH(Sinopharm Chemical Reagent Co.,目录号:10019718)
  11. 丙酮(100%)(Sigma-Aldrich,目录号:69508)
  12. 乙醇(100%)(Sinopharm Chemical Reagent Co.,目录号:100092690)
  13. 乙二醇-O,O'-双(2-氨基乙基)-1 N,N,N',N'-四乙酸(EGTA)(Sigma-Aldrich,目录号:E3889)
  14. 1M HCl(Sinopharm Chemical Reagent Co.,目录号:10011061)
  15. Epon 812(Sigma-Aldrich,目录号:45346)
  16. Formvar溶液(Sigma-Aldrich,目录号:09823)

设备

  1. 冰箱(4°C)
  2. 孵化器
  3. 真空设备(真空泵连接到干燥器)
  4. pH计
  5. 透射电子显微镜(Hitachi High-Technologies Europe GmbH,型号:H-7500)
  6. 超微切片机(Leica Microsystems,型号:EM UC6)
  7. 镊子(尖头)
  8. 玻璃烧杯
  9. 玻璃搅拌棒

程序

  1. 制备PPA染色溶液
    1. 制备4%PPA溶液:溶解8.0g焦磷酸钾(PPA) ?通过加热至90-95℃并且连续地加入400mM Na 2 HPO 4(pH 7.6) 搅动。然后,通过0.22μmMillipore过滤溶液 过滤器,然后在使用前保存在4°C冰箱中(见注1)
    2. 制备PPA染色液(50ml):混合25ml 4%PPA溶液与6 ml 25%戊二醛,然后用19ml无菌H 2 O填充(参见注释 ?2)。
  2. 固定和漂洗:收集生长在1/2 MS培养基上的植物根尖,并用蒸馏水冲洗数次,然后用尺寸为约2mm的手术刀切割裂口尖端,并迅速将其置于1.5ml离心机中的冷PPA染色液中管。如果植物材料漂浮,进行真空处理(0.8Mpa,10分钟),直到材料沉到管的底部,在4℃固定,过夜。弃去PPA染色液,用1%PPA溶液冲洗3次,每次5分钟
  3. 脱水:然后,将固定材料在分级的冷丙酮系列(30%,50%,70%,90%,100%,100%)中脱水,每次15分钟。
  4. 渗透:脱水后,用Epon 812:纯丙酮(1:3,1:1和3:1)的系列混合物处理材料,每次12小时;和纯Epon 812混合物(35-40℃)12小时
  5. 嵌入:添加新鲜的Epon 812混合物以清洁200微升eppendorf管,小心地转移根尖,组织根尖,并让其合并在包埋混合物中,但不能用针接触管壁(图1A)。然后将管置于培养箱中,35℃12小时,然后45℃12小时和60℃24小时,直到树脂良好固化。在室温下冷却约1小时。
  6. 涂层镍网:可以通过将网格在formvar溶液中合并几秒钟,然后在滤纸上干燥约1至2小时来完成具有formvar的涂层镍网格;或将一滴formvar溶液直接放在干净的草坪上的镍网上,然后让其自然干燥,约需2小时。
  7. 切片:在用锋利的刀(或块修剪器)适当地修剪嵌入块之后,用超切片切片机切割该块以产生100nm厚的超薄切片,并安装到200目的形成有镀层的镍网上(图1B) 。在灯下干燥约1小时。
  8. 显微镜:用透射电子显微镜检查样品。钙沉积显示黑色颗粒或聚集体(图1C-D;注释3)

    图1.苜蓿根尖细胞中的取样和钙沉积的代表性模式。 A.嵌入混合物中样品的图示; B.镍网格样品截面图。 C. rstB 转基因苜蓿的皮层细胞壁显示大的聚集物沉积在膜和细胞质的残余物上。 wt植物的表皮细胞壁显示弱的钙染色。沉积的颗粒较小。用固体红色箭头指示钙沉积。脂质体(液滴)用蓝色虚线箭头表示。刻度条代表2μm。

笔记

  1. 对于植物细胞固定,戊二醛的浓度可以为2.5%-4%。在该方案中,使用3.0%Glutaraldehye
  2. 对于钙染色,在不同方案中PPA浓度可以为0.5%至2.5%
  3. 为了证实所观察到的颗粒是钙沉积物,可以在60℃下用0.1M EGTA(pH 8.0)处理切片30分钟,然后再次检查。钙颗粒可以通过EGTA处理除去(Stockwell和Hanchey,1982)

食谱

  1. 1 H Na 2 HPO 4(pH 7.6) 在1L水中溶解138g NaH 2 PO 4 PO 4,H 2 H 2 O并溶解142g Na 2 SO 4,在1L水中制备它们的1M储备溶液。
    HPO 在此期间,用NaOH或HCl测试并改变pH值至7.6
  2. 400mM Na 2 HPO 4(pH 7.6)
    将338ml 1M Na 2 HPO 4和62ml 1M NaH 2 PO 4溶液混合,并加入水至最终1 L
  3. 200mM Na 2 HPO 4(pH 7.6)
    用等体积的蒸馏水稀释400mM Na 2 HPO 4水溶液
  4. 4%PPA溶液
    在发烟窗中,通过加热至90-95℃并在连续搅拌下将8.0g焦锑酸钾(PPA)溶解在400mM Na 2 HPO 4(pH 7.6)中。
  5. PPA染色液(50ml) 将4%PPA溶液25ml,25%戊二醛6ml和19ml无菌H 2 O混合物混合
  6. 0.1 M EGTA
    在约80ml水中加入18.61g EGTA,2.0g NaOH,继续搅拌直至完全溶解
    加水至100 ml
    将pH值调整为8.0
  7. 1 M NaOH
    将4.0g NaOH溶于100ml水中
  8. 1 M HCl
    取8.36ml 37%HCl,加水至100ml

致谢

该方案是来自Stockwell和Hanchey(1982)和Borgers等人(1982)的改编。这项工作得到了中国国家自然科学基金(31472140)的支持。我们感谢Hao女士在钙定位测试中的技术援助。

参考文献

  1. Borgers,M.,Thone,F.,Verheyen,A.and Ter Keurs,H.E。(1984)。 钙在骨骼和心肌中的定位。 Histochem J 16(3):295-309。
  2. Stockwell,V.and Hanchey,P。(1982)。 钙定位的细胞组织化学技术及其在病变植物中的应用植物生理 70(1):244-251。
  3. Wick,S.M。和Hepler,P.K。(1982)。 使用锑酸钾选择性定位细胞内Ca 2+ 2 + 。 J Histochem Cytochem 30(11):1190-1204。
  4. Zhang,W. J.和Wang,T。(2015)。 通过rstB基因转化提高苜蓿(苜蓿)的耐盐性。 Plant Sci 234:110-118。
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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
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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