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Aniline Blue and Calcofluor White Staining of Callose and Cellulose in the Streptophyte Green Algae Zygnema and Klebsormidium
采用苯胺蓝和Calcofluor White对绿藻类双星藻属和克里藻属的胼胝质和纤维素进行染色   

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Abstract

Plant including green algal cells are surrounded by a cell wall, which is a diverse composite of complex polysaccharides and crucial for their function and survival. Here we describe two simple protocols to visualize callose (1→3-β-D-glucose) and cellulose (1→4-β-D-glucose) and related polysaccharides in the cell walls of streptophyte green algae. Untreated or algal cells heated in NaOH are incubated in Calcofluor white (binding to β-glucans including cellulose) or Aniline blue (binding to callose), respectively. Both dyes can be visualized by epifluorescence microscopy.

Background

Due to its easy and quick applicability, Aniline blue was used to visualize callose in various strains of Klebsormidium sp. and Zygnema sp., before more laborious fixation and immunolocalisation protocols were applied (Herburger and Holzinger, 2015). Applying Aniline blue staining and monoclonal antibodies against callose brought similar results (Herburger and Holzinger, 2015). Calcofluor white staining is the fastest way to visualize the 1→4-β-glucan fraction including cellulose of the cell wall, since no pre-treatments are required.

Materials and Reagents

  1. 2 ml Eppendorf tubes
  2. Epoxy coated microscopic slides with 8-12 wells (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1014326410EPOXY ) and cover slips
  3. Algal cells or filaments (Figure 1A)
    Note: In principle, this protocol is not restricted to green algae and can be applied to all plant cells surrounded by a cell wall, including cell cultures.
  4. Sodium hydroxide pellets (NaOH) (EMD Millipore, catalog number: 106469 )
  5. A. bidest. (double distilled water)
  6. Sodium phosphate monobasic dihydrate (NaH2PO4·2H2O) (EMD Millipore, catalog number: 1063420250 )
  7. Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (EMD Millipore, catalog number: 1065800500 )
  8. Culture medium (BBM [Bischoff and Bold, 1963], MBBM [Starr and Zeikus, 1993])
  9. Aniline blue diammonium salt (Sigma-Aldrich, catalog number: 415049 )
  10. Calcofluor white (Sigma-Aldrich, catalog number: 18909 )
    Note: Adding Evans blue as a counterstain to diminish background fluorescence is not obligatory, since background fluorescence in the algae investigated is very low.
  11. 0.2 M NaH2PO4·2H2O stock solution (see Recipes)
  12. 0.2 M Na2HPO4·2H2O stock solution (see Recipes)
  13. 50 ml of Sørensen’s phosphate buffer (see Recipes)

Equipment

  1. Glass Pasteur pipettes
  2. 25 ml beaker
  3. 1,000 ml and 500 ml volumetric flask
  4. 100 ml Schott flask
  5. Balance
  6. Pair of fine-pointed tweezers
  7. Metal rack (1.5 ml microfuge rack) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3166185 )
  8. Water bath or heat plate and beaker
  9. Tabletop centrifuge (Sigma Laborzentrifugen, model: Sigma 1-14 )
  10. Shaker (Thermo Fisher Scientific, Thermo ScientificTM, model: Compact Digital Microplate Shaker)
  11. Epifluorescence microscope capable of UV excitation and long pass detection, with high numerical aperture lens, connected to a camera for documentation (e.g., We used a ZEISS Axiovert 200M equipped with a 63 x 1.4 NA objective, a OSRAM HBO 50 Q/AC L1 CZ Mercury short ARC Photo optic lamp, Zeiss Filter Set 01 (excitation: band pass [BP] 365/12 nm; emission: long pass [LP] 397 nm and connected to a Axiocam MRc5 camera) (Carl Zeiss, model: Axiovert 200M )

Procedure

  1. Aniline blue staining
    1. Prepare 20 ml of 8 N NaOH from sodium hydroxide pellets in double distilled water (A. bidest.) in a 25 ml beaker.
    2. Prepare 50 ml of Sørensen’s phosphate buffer (0.1 M, pH = 8.0).
    3. Transfer algae to 2 ml Eppendorf tubes (Figure 1B) and wash them once with culture medium.
      Note: Washing can be omitted if algae from lab cultures are investigated. Avoid mechanical stress or damage to the cells by using a pair of fine-pointed tweezers. This prevents additional short-term incorporation of callose in the cell walls. Single-celled organisms or cells surrounded by a sticky mucilage layer can be transferred easily by using glass pipettes.
    4. Decant and discard culture medium and add 1.5 ml 8 N NaOH in the tube (Figure 1C).
      Note: Centrifuge at ~1,000 x g to sediment biomass in order to remove liquid.
    5. Incubate the tubes at 60 °C for 20 min.
      Note: An easy way to do this is fixing the tubes in a metal rack in a pre-heated water bath or in a beaker on a heat plate. Maintain bath level at same level as the liquid in the tubes.
    6. Decant and discard the 8 N NaOH and wash algae 3 x with A. bidest. for at least 5 min.
    7. Add 1% aniline blue (w/v) to freshly prepared Sørensen’s phosphate buffer.
    8. In the following, it is recommended to work under low light intensities (~5 μmol photons m-2 s-1) to prevent degradation of the dye.
    9. Stain algae in 1.5 ml of 1% aniline blue for 1 h (Figure 1D).
      Note: Incubate in darkness under gentle shaking.
    10. Wash algae 2 x with Sørensen’s phosphate buffer for 5 min and transfer to microscopic slides (mount in Sørensen’s phosphate buffer).
    11. Visualize aniline blue with an epifluorescence microscope (excitation: band pass [BP] 365/12 nm; emission: long pass [LP] 397 nm; Figure 1E).
      Note: Aniline blue staining allows a quick and easy-to-use visualization of callose in plant’s cell walls. Furthermore, Aniline blue can be used to quantify the callose content in plant material spectrofluorimetrically (Köhle et al., 1985). For a more specific detection of callose at the confocal laser scanning and transmission electron microscopy level, commercially available monoclonal antibodies can be applied (Meikle et al., 1991; Herburger and Holzinger, 2015).

  2. Calcofluor white staining
    1. Add 1% (v/v) Calcofluor white to 50 ml of A. dest.
    2. Transfer algae to 2 ml Eppendorf tubes and wash once with culture medium (Figure 1B).
      Note: Washing can be omitted, if algal cultures are investigated. Single-celled organisms or cells surrounded by a sticky mucilage layer can be transferred easily by using glass pipettes.
    3. Stain algae with 1.5 ml 1% Calcofluor white for 20 sec (Figure 1F).
      Note: Krishnamurthy (1999) describes protocols using 0.01-0.7% (v/v) Calcofluor white in buffered or unbuffered solutions and incubation times up to 2 min. Although our plant material was rather small (algal filaments with a max. diameter of ~24 µm), a 1% aqueous solution of Calcofluor white produced the best results. Lower concentrations yielded very low fluorescence signals despite incubation times up to 10 min.
    4. Wash algae twice in A. dest. for 30 sec and transfer immediately to microscopic slides (mount in A. dest.).
      Note: To immobilize entire algal filaments in one optical plane, epoxy-coated microscopic slides can be used. This allows focusing the central area of algal filaments and reduces blur effects by filament’s top or bottom side (Figure 1G).
    5. Visualize Calcofluor white with an epifluoresence microscope (excitation: band pass [BP] 365/12 nm; emission: long pass [LP] 397 nm; Figure 1G).
      Note: Excitation at 400-410 is also fine. Calcofluor white may not specifically bind to cellulose and can also stain callose, chitin, mixed-linkage glycans or other cell wall polysaccharides (Krishnamurthy, 1999).


      Figure 1. (A-G) Summary of the experimental procedure for Aniline blue (C-E) and Calcofluor white (F, G) staining. Aniline blue (E) and Calcofluor white (G) fluorescence is shown in blue. Bright field and corresponding fluorescence images in (E) and (G) are reprinted from Herburger and Holzinger (2015). Scale bars = 10 µm.

Recipes

  1. 0.2 M NaH2PO4·2H2O stock solution
    Add 15.60 g sodium phosphate monobasic dehydrate (NaH2PO4·2H2O) to 500 ml of A. bidest.
    Note: Can be stored at 4 °C for several months.
  2. 0.2 M Na2HPO4·2H2O stock solution
    Add 35.61 g sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) to 1,000 ml of A. bidest.
    Note: Can be stored at 4 °C for several months.
  3. 50 ml of Sørensen’s phosphate buffer (0.1 M, pH = 8.0)
    Mix 2.7 ml of 0.2 M NaH2PO4·2H2O stock solution with 47.3 ml of 0.2 M Na2HPO4·2H2O stock solution (Sørensen, 1909) in a 100 ml Schott flask.

Acknowledgments

Aniline blue staining as applied by Herburger and Holzinger (2015) and described in this protocol is based on the fluorescence method of Currier et al. (1955), previously used for pollen tube staining (Linskens and Esser, 1957). Calcofluor white staining follows the protocol of Krishnamurthy (1999). The authors declare that there is no conflict of interests. The study was funded by Austrian Science Fund (FWF) projects P 24242-B16 and I 1951-B16 to A.H.

References

  1. Bischoff, H. W. and Bold, H. C. (1963). Phycological studies IV. Some soil algae from Enchanted Rock and related algal species. Univ Tex Publ 6318: 1-95.
  2. Currier, H. B., Esau, K. and Cheadle, V. I. (1955). Plasmolytic studies of phloem. Am J Bot 42: 68-81.
  3. Herburger, K. and Holzinger, A. (2015). Localization and quantification of callose in the streptophyte green algae Zygnema and Klebsormidium: Correlation with desiccation tolerance. Plant Cell Physiol 56(11): 2259-2270.
  4. Krishnamurthy, K. V. (1999). Methods in cell wall cytochemistry. CRC Press: 156-158.
  5. Köhle, H., Jeblick, W., Poten, F., Blaschek, W. and Kauss, H. (1985). Chitosan-elicited callose synthesis in soybean cells as a ca-dependent process. Plant Physiol 77(3): 544-551.
  6. Linskens, H. F. and Esser, K. L. (1957). Über eine spezifische Anfärbung der Pollenschläuche im Griffel und die Zahl der Kallosepfropfen nach Selbstung und Fremdung. Naturwissenschaften 44: 16-16.
  7. Meikle, P. J., Bonig, I., Hoogenraad, N. J., Clarke, A. E. and Stone, B. A. (1991). The location of (1-->3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1-->3)-β-glucan-specific monoclonal antibody. Planta 185(1): 1-8.
  8. Sørenson, S. P. L. (1909). Enzymstudien. II, Über die Messung und die Bedeutung der Wasserstoffionenkonzentration bei enzymatischen Prozessen. Biochem Zeitschr 21: 131-304.
  9. Starr, R. C. and Zeikus, J. A. (1993). UTEX - the culture collection of algae atthe University of Texas at Austin 1993 list of cultures. J Phycol 29: 1-106.

简介

包括绿藻细胞的植物被细胞壁包围,所述细胞壁是复合多糖的多样复合物,并且对于它们的功能和存活是至关重要的。在这里我们描述两个简单的协议,可视化胼lose质(1→3-β-D-葡萄糖)和纤维素(1→4-β-D-葡萄糖)和相关的多糖在链霉素绿藻的细胞壁。将在NaOH中加热的未处理或藻类细胞分别在Calcofluor白(结合β-葡聚糖包括纤维素)或苯胺蓝(结合胼cal质)中孵育。两种染料都可以通过落射荧光显微镜观察。

[背景] 由于其容易和快速的适用性,使用苯胺蓝显现各种株Klebsormidium sp中的胼。质。和在更艰苦的固定和免疫定位方案(Herburger和Holzinger,2015)之前使用Zygnema。应用苯胺蓝染色和单克隆抗体对胼lose质产生类似的结果(Herburger和Holzinger,2015)。 Calcofluor白色染色是观察包括细胞壁的纤维素的1→4-β-葡聚糖部分的最快方式,因为不需要预处理。

材料和试剂

  1. 2 ml Eppendorf管
  2. 具有8-12个孔的环氧涂覆的显微镜载玻片(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:1014326410EPOXY)和盖玻片
  3. 藻细胞或细丝(图1A)
    注意:原则上,此协议不限于绿藻,可应用于细胞壁包围的所有植物细胞,包括细胞培养物。
  4. 氢氧化钠丸粒(NaOH)(EMD Millipore,目录号:106469)
  5. A. bidest。 (双蒸馏水)
  6. 磷酸二氢钠二水合物(NaH 2 PO 4·2H 2 O)(EMD Millipore,目录号:1063420250)
  7. 磷酸氢二钠二水合物(Na 2 HPO 4·2H 2 O)(EMD Millipore,目录号:1065800500)
  8. 培养基(BBM [Bischoff和Bold,1963],MBBM [Starr和Zeikus,1993]) sup>
  9. 苯胺蓝二铵盐(Sigma-Aldrich,目录号:415049)
  10. Calcofluor白(Sigma-Aldrich,目录号:18909)
    注意:添加伊文思蓝作为复染剂以减少背景荧光并不是必须的,因为所研究的藻类中的背景荧光非常低。
  11. 0.2M NaH 2 PO 4/2H 2 O储备溶液(参见配方)
  12. 0.2M Na 2 HPO 4·2H 2 O储备溶液(参见配方)。
  13. 50mlS?rensen磷酸盐缓冲液(见配方)

设备

  1. 玻璃巴斯德移液器
  2. 25 ml烧杯
  3. 1,000ml和500ml容量瓶
  4. 100 ml Schott培养瓶
  5. 余额
  6. 一对细尖镊子
  7. 金属架(1.5ml微量离心机架)(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:3166185)
  8. 水浴或加热板和烧杯
  9. 台式离心机(Sigma Laborzentrifugen,型号:Sigma 1-14)
  10. Shaker(Thermo Fisher Scientific,Thermo Scientific TM ,型号:Compact Digital Microplate Shaker)
  11. 具有UV激发和长通检测的荧光显微镜,具有高数值孔径透镜,连接到相机用于记录(例如。),我们使用配备有63×1.4NA物镜的蔡司Axiovert 200M, OSRAM HBO 50 Q/AC L1 CZ汞短光ARC光学灯Zeiss Filter Set 01(激发:带通[BP] 365/12 nm;发射:长通[LP] 397 nm并连接到Axiocam MRc5相机) Carl Zeiss,型号:Axiovert 200M)

程序

  1. 苯胺蓝染色
    1. 在25ml烧杯中,从双蒸水(A. bidest)中的氢氧化钠颗粒中制备20ml 8N NaOH。
    2. 准备50mlS?rensen的磷酸盐缓冲液(0.1M,pH = 8.0)
    3. 转移藻类到2毫升Eppendorf管(图1B),并用培养基洗涤一次 注意:如果研究来自实验室培养物的藻类,可以省略洗涤。通过使用一对细尖镊子避免机械应力或细胞损伤。这防止胼prevents质在细胞壁中的额外短期掺入。单细胞生物体或由粘性粘液层包围的细胞可以通过使用玻璃移液管轻松转移。
    4. 倾析并弃去培养基,并在管中加入1.5ml 8N NaOH(图1C) 注意:以约1,000 x g离心以沉淀生物质以清除液体。
    5. 将管在60℃孵育20分钟。
      注意:一个简单的方法是将管子固定在预热水浴中的金属架上或加热板上的烧杯中。保持浴液水平与管中液体的水平相同。
    6. 滗析并丢弃8 N NaOH,用A. bidest洗涤藻类3次。至少5分钟。
    7. 向新鲜制备的S?rensen磷酸盐缓冲液中加入1%苯胺蓝(w/v)
    8. 在下文中,建议在低光强度(?5μmol光子m <-200/s -1 )下工作以防止染料降解。
    9. 在1.5ml的1%苯胺蓝中染色藻1小时(图1D) 注意:在温和的摇晃下在黑暗中孵育。
    10. 用S?rensen的磷酸盐缓冲液洗涤藻类2次,每次5分钟,转移到显微镜载玻片(装在S?rensen的磷酸盐缓冲液中)。
    11. 用荧光显微镜观察苯胺蓝(激发:带通[BP] 365/12nm;发射:长通[LP] 397nm;图1E)。 注意:苯胺蓝染色允许在植物细胞壁中快速且易于使用胼。质的可视化。此外,苯胺蓝可用于定量分析荧光分析的植物材料中的胼lose质含量(K?hle等人,1985)。为了在共聚焦激光扫描和透射电子显微镜水平更具体地检测胼cal质,可以应用市售的单克隆抗体(Meikle等人,1991; Herburger和Holzinger,2015)。
  2. Calcofluor白色染色
    1. 将1%(v/v)Calcofluor白色加入到50ml目的物中
    2. 转移藻类到2毫升Eppendorf管,用培养基洗一次(图1B) 注意:如果研究藻类培养物,可以省略洗涤。单细胞生物体或由粘性粘液层包围的细胞可以通过使用玻璃移液管容易地转移。
    3. 用1.5ml 1%Calcofluor白色染色20秒(图1F) 注意:Krishnamurthy(1999)描述了在缓冲或未缓冲的溶液中使用0.01-0.7%(v/v)Calcofluor白色的方案以及高达2分钟的孵育时间。虽然我们的植物材料相当小(藻丝最大直径?24μm),1%的Calcofluor白水溶液产生最好的结果。较低的浓度产生非常低的荧光信号,尽管孵育时间高达10分钟。
    4. 在A. dest中洗涤藻类两次。 30秒,并立即转移到显微镜载玻片(安装在目标)。
      注意:为了将整个藻类细丝固定在一个光学平面中,可以使用环氧涂覆的微观载玻片。这允许聚焦藻类细丝的中心区域,并减少细丝顶部或底部的模糊效果(图片 1G)。
    5. 使用落射荧光显微镜(激发:带通[BP] 365/12nm;发射:长通[LP] 397nm;图1G)显现Calcofluor白色。 注意:在400-410的激励也很好。 Calcofluor白色可能不特异性结合纤维素,也可以染色胼cal质,壳多糖,混合连接聚糖或其他细胞壁多糖(Krishnamurthy,1999)。


      图1.(AG)苯胺蓝(CE)和Calcofluor白(F,G)染色的实验程序的总结。苯胺蓝(E)和Calcofluor白(G)荧光以蓝色显示。 (E)和(G)中的明场和相应的荧光图像由Herburger和Holzinger(2015)重印。比例尺=10μm

食谱

  1. 0.2M NaH 2 PO 4 PO 4·2H 2 O储备溶液
    将15.60g磷酸二氢钠一水合物(NaH 2 PO 4·2H 2 O)加入到500ml的丙二醇中。
    注意:可以在4°C储存数个月。
  2. 0.2M Na 2 HPO 4·2H 2 O储备溶液。
    将35.61g磷酸氢二钠二水合物(Na 2 HPO 4·2H 2 O)加入到1,000ml的丙二醇中。
    注意:可以在4°C储存数个月。
  3. 50mlS?rensen磷酸盐缓冲液(0.1M,pH = 8.0) 将2.7ml的0.2M NaH 2 PO 4·2H 2 O储备溶液与47.3ml的0.2M Na 2 SO 4溶液混合,在100毫升Schott烧瓶中加入HPO 4 H 2 O 2储备溶液(S?rensen,1909)。

致谢

由Herburger和Holzinger(2015)应用并在该方案中描述的苯胺蓝染色基于Currier等人的荧光方法。 (1955),以前用于花粉管染色(Linskens和Esser,1957)。 Calcofluor白色染色遵循Krishnamurthy(1999)的方案。作者声明,没有利益冲突。该研究由奥地利科学基金(FWF)项目P24242-B16和I1951-B16授予A.H.

参考文献

  1. Bischoff,H. W. and Bold,H. C.(1963)。  真菌学研究IV。来自Enchanted Rock的一些土壤藻类和相关的藻类物种。 Univ Tex Publ 6318:1-95。
  2. Currier,HB,Esau,K. and Cheadle,VI(1955)。  韧皮部的溶血研究。 42:68-81。
  3. Herburger,K.和Holzinger,A。(2015)。  链霉菌绿藻中的胼>质的定位和定量 :与干燥耐受性的相关性 植物细胞生理< 11):2259-2270
  4. Krishnamurthy,KV(1999)。  细胞壁细胞化学方法。 CRC :156-158。
  5. K?hle,H.,Jeblick,W.,Poten,F.,Blaschek,W.and Kauss,H。(1985)。  壳聚糖 - 引起大豆细胞中的胼cal质合成作为ca-依赖性过程。植物生理学77(3):544 -551。
  6. Linskens,HF和Esser,KL(1957)。  übereine spezifische Naturwissenschaften 44:16-16。< br>< br /
  7. Meikle,PJ,Bonig,I.,Hoogenraad,NJ,Clarke,AEand Stone,BA(1991)。  (1→3)-β-葡聚糖在烟草花粉的花粉壁中的位置使用(1→3) ; 3)-β-葡聚糖特异性单克隆抗体 185(1):1-8。
  8. S?renson,SPL(1909)。  Enzymstudien。 II,überdie Messung und die Bedeutung der Wasserstoffionenkonzentration bei enzymatischen Prozessen。 Biochem Zeitschr 21:131-304。
  9. Starr,RC和Zeikus,JA(1993)。  UTEX - 德克萨斯大学奥斯汀分校1993年的藻类文化收藏1993年的文化名单 J Phycol 29:1-106。
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引用:Herburger, K. and Holzinger, A. (2016). Aniline Blue and Calcofluor White Staining of Callose and Cellulose in the Streptophyte Green Algae Zygnema and Klebsormidium. Bio-protocol 6(20): e1969. DOI: 10.21769/BioProtoc.1969.
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