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Immunoelectron Microscopy in Chlamydomonas Cells
免疫电镜术检测衣藻属细胞   

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Abstract

The method of immunoelectron microscopy is intended for localization of proteins inside the cells of Chlamydomonas reinhardtii or other microalgae and cyanobacteria. This protocol was used to study localization of carbonic anhydrase Cah3 with antibodies raised in rabbit, though it can be used to localize any other abundant protein. Primary rabbit antibodies are recommended because they react quickly and specifically with proteins of C. reinhardtii. If primary antibodies other than rabbit are used, the blocking procedure and time of incubation with primary and secondary antibodies should be adjusted.

Keywords: Ultrastructure(超微结构), Chlamydomonas reinhardtii(莱茵衣藻), Carbonic anhydrase(碳酸酐酶), Localization(定位)

Materials and Reagents

  1. Culture of C. reinhardtii 137mt+ (WT) (we used the strain IPPAS D-298 from the Collection of microalgae of the Institute of Plant Physiology RAS, Moscow)
  2. Primary antibodies raised in rabbits against the protein of interest (we used antibodies raised against the recombinant Cah-3 protein (α-CA) of C. reinhardtii, Agrisera, Vannas, Sweden)
  3. Secondary antibodies: Anti-Rabbit IgG conjugated with 10 nm colloidal gold particles (Sigma-Aldrich, catalog number: G7402 )
  4. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  5. LR White embedding kit (Sigma-Aldrich, catalog number: 62662-1EA-F )
  6. Gelatin capsules (SPI supplies, catalog number: 02308-SS )
  7. Formvar and carbon-coated nickel grids (SPI supplies, catalog number: 3430N-CF )
  8. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A7906 )
  9. Goat serum (Sigma-Aldrich, catalog number: G9023 )
  10. Rabbit serum (Sigma-Aldrich, catalog number: R4505 )
  11. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148)
  12. Ethanol
    Optional
  13. Uranyl acetate (e.g. SPI supplies, catalog number: 02624-AB )
  14. Lead citrate (e.g. Sigma-Aldrich, catalog number: 15326 )
  15. NaOH
  16. Phosphate buffer saline (PBS) (pH 7.4) (see Recipes)
  17. Fixation solution (4% paraformaldehyde) (see Recipes)
  18. Tris-buffer saline (TBS) (pH 7.4) (see Recipes)
  19. 1% BSA-TBS (see Recipes)
  20. Uranyl acetate (see Recipes)
  21. Lead citrate (see Recipes)

Equipment

  1. 96 well immunology plate (e.g. Greiner Bio-One, catalog number: 650001 )
  2. Pasteur pipettes with thin tips (ROTHE 306.1)
  3. Thermostat unit with, at least, 37-55 °C temperature range (e.g. BINDER B28)
  4. Ultramicrotome (e.g. Reichert, OMU-3, Austria)
  5. Grid storage box (Sigma-Aldrich, catalog number: G6276 )
  6. Transmission electron microscope (e.g. Libra-120, Carl Zeiss, Germany)
  7. Plastic Petri dish or Teflon plate with hydrophobic surface
  8. Wet chamber

Procedure

  1. Sample preparation:
    Spin down the samples of C. reinhardtii culture in 2 ml Eppendorf tubes at 13,000 x g at room temperature for 5 min. The optimal volume of the obtained pellet, which would be sufficient for embedding in 2-3 gelatin capsules, is about 50-75 μl.
  2. Fixation:
    Remove supernatant and add 0.5 ml of fixation solution (4% paraformaldehyde). For proper fixation, the minimum incubation time is 3 h at 4 °C.
    Note: Duration of fixation can be prolonged up to several days. This fixation solution preserves membranes and most proteins, but it may dislocate starch and proteins after several weeks of storage.
  3. Embedding (all steps are carried out at room temperature):
    1. Wash the pellet with PBS buffer in the same Eppendorf tube:
      Remove supernatant with pipette.
      Add 1 ml of PBS buffer and vortex it gently.
      Incubate for 5 min.
      Spin down as described in step 1.
      Repeat this washing step 3 times.
    2. Dehydrate pellet with series of ethanol dilution, twice in each concentration of ethanol:
      20% (v/v) 15 min + 15 min
      40% (v/v) 15 min + 15 min
      50% (v/v) 20 min + 20 min
      70% (v/v) 20 min + 20 min
      For each dehydration step, discard previous solution and add 0.5 ml of the next sequential solution with clean pipette. Incubate for the indicated time. We recommend using freshly prepared solutions pre-chilled at 4 °C. Take care not to touch or disturb the pellet during these steps.
      Note: It is handy to use a disposable plastic Pasteur pipette with a thin tip to discard the solutions. If the pellet is disturbed, centrifuge the Eppendorf tube as described in step 1. If it is necessary to interrupt the procedure at this step, the samples can be stored in 70% ethanol overnight at 4 °C.
    3. Infiltration: Sequentially replace 70% ethanol with the mixture of 70% ethanol and LR White resin:
      3:1 70% ethanol: LR White for 1 h
      1:1 70% ethanol: LR White for 1 h
      1:3 70% ethanol: LR White for 1 h
      Next steps require the complete replacement of ethanol with LR White resin:
      Replace the 1:3 ethanol/resin mixture with LR White resin alone for 1 h.
      Replace it again with fresh portion of the LR White resin and leave overnight.
      Next day replace it with new portion of the LR White resin and keep it for embedding.
      Note: LR White resin should be mixed with catalyst according to manufacturer’s protocol for 24 h before use. With time, a resin-ethanol mixture may stratify into layers. If this happens, mix the layers gently several times with a pipette (care should be taken not to damage the pellet).
    4. Embedding:
      Put gelatin capsules into wells of 96-wells immunology plate.
      Add one drop of the resin into each gelatin capsule.
      Put a small piece of the pellet into the capsule – one pellet usually goes into 2-3 capsules.
      Fill up capsules with resin. Avoid overflow. Close capsules carefully with their caps.
      Incubate at 37 °C for 1-4 days (or at 45 °C for one day) and then at 55 °C until polymerization is completed (usually 1-3 days).
      Note: Longer incubation at lower temperature helps to receive softer block, shorter incubation at higher temperature helps to receive firmer block, but more fragile. Completeness of polymerization can be checked with a needle: The resin should be solid but not fragile.
    5. Cut the sample into sections with a microtome and place them onto Formvar and carbon-coated nickel grids.
      Note: Formvar+carbon coating prevents unspecific labeling and improves the quality of sections (without carbon layer sections often get wrinkled).
  4. Immunocytochemistry
    Carry out all of the following manipulations with grids on the surface of 50-100 μl drops on a hydrophobic surface (e.g. plastic Petri dish or Teflon plate).
    A tip: For better interaction of grid surfaces with solution, put the hydrophobic surface with drops and grids on a table that is slightly trembling due to vibration of a vortex. Adjust the intensity of the vibrations so that trembling of the drop surfaces would be noticeable.
    When overnight incubation of the grids is required, put them into a wet chamber at 4 °C. Do not allow grids to dry out during next steps.
    1. Block nonspecific interaction of antibodies on a drop of 20% goat serum in BSA-TBS for 1 h.
      Note: If nonspecific labeling still happens, increase the proportion of the goat serum up to 50% and BSA up to 5%.
    2. Put the grids onto the 100 μl drops with properly diluted primary antibodies raised against the protein of interest. The dilution can be determined only empirically; usually it is in range of 1:16 – 1:128.
      For negative control, apply rabbit serum or, better, the pre-immune serum instead of primary antibodies to the target protein.
      Add purified protein of interest to solution of primary antibodies as positive control.
      Leave grids overnight at 4 °C. You may vary time and temperature to increase or decrease the efficiency of binding and the intensity of labeling.
    3. Wash grids on drops with TBS 5 times, leaving them on drops for 5-10 min between changes of TBS solution.
    4. Transfer grids onto 100 μl drops of properly diluted (according to the protocol of manufacturer) Anti-Rabbit IgG conjugated with 10 nm colloidal gold particles in BSA-TBS and expose them for 1 h at room temperature.
    5. Wash grids on drops with TBS 5 times, then with deionized water. These washing steps should be done by quick repeated submersion of grids into 3-5 ml of TBS and water during 1 min.
    6. Dry grids on filter paper and put them into grid storage box.
    7. Optional – sequentially contrast the grids with uranyl acetate and lead citrate. Prepare wet chamber with NaOH crystals and filter paper wetted with 0.2 M NaOH (Figure 1). Put grids onto 100 μl drops of uranyl acetate for 45-60 min. Pour 50 μl drops of lead citrate onto the same plate. Wash grids by quick repeated submersion first into 3-5 ml of 0.2 M NaOH (5-10 times), and then – into 3-5 ml of boiled deionized water (for 1 min). NaOH solution and boiled water are needed to avoid the reaction of CO2 with lead, which results in formation of insoluble crystals. Put grids onto prepared lead citrate drops for 5-7 min. Wash in the same way as performed after uranyl acetate. Dry grids on filter paper and put them into grid storage box.
  5. Analysis of sections with electron transmission microscope.
    Note: Only a few gold particles are allowed to be as a background in negative and positive controls.


    Figure 1. Wet chamber with NaOH crystals for the contrast staining

Recipes

  1. Phosphate buffer saline (PBS) (pH 7.4)
    1. Prepare stock solutions:
      Stock 1: NaH2PO4 2.76 g per 100 ml
      Stock 2: Na2HPO4 14.2 g per 500 ml
    2. For 0.5 L of PBS buffer mix 95 ml of the Stock 1 and 405 ml of the stock 2 and add
      4.25 g of NaCl (final concentration is 0.85%)
      pH of this solution should be 7.4 (may be adjusted with NaOH)
  2. Fixation solution (4% paraformaldehyde)
    1. Heat 50 ml of PBS (pH 7.4) to 70 °C
    2. Dissolve 2 g of paraformaldehyde with stirring in hot PBS.
    3. Cool the solution at room temperature and filter through a filter paper
      (Keep the solution at 4 °C in darkness, use within 1 week)
  3. Tris-buffer saline (TBS) (pH 7.4)
    Tris-HCl 2.4 g/L
    NaCl 8.76 g/L
    Adjust pH to 7.4 with HCl
  4. 1% BSA-TBS
    Add 10 g/L of BSA to TBS
    Keep aliquots frozen at -20 °C, or use fresh.
  5. Uranyl acetate
    Prepare a saturated solution of uranyl acetate in 50% ethanol. Filter it through a filter paper. Use fresh.
  6. Lead citrate
    1. Dissolve 1.33 g Pb(NO3)2 and 1.76 g Na3(C6H5O7).2H2O in 30 ml of deionized water. Shake it for 30 min in the orbital shaker. At this stage the solution may appear milky.
    2. Add 8 ml of 1 M NaOH: the solution should become transparent. Adjust the volume to 50 ml with deionized water. pH should be about 12.
      Keep in small aliquots at -20 °C. Thaw and spin down before use.

Acknowledgments

This protocol was adapted from Sinetova et al. (2012). We appreciate Prof. J. Moroney (Louisiana State University, USA) for providing the strain of C. reinhardtii 137mt+ and CCM-mutant cia3, Prof. G. Samuelsson (Umea University, Sweden) who kindly supplied us with primary antibodies against recombinant Cah3 and Cah3-MBP fusion protein. We thank Prof. Pronina N.A. and Dr. Kupriyanova E.V. for the physiological and molecular biological base of this protocol. This work was supported by a grant from the Russian Foundation for Basic Research (no. 10-04-01463), a grant from the Presidium of Russian Academy of Sciences (program “Molecular and Cell Biology”), and a grant from the Russian Ministry of Education and Science (no. 16.740.11.0176).

References

  1. Oliver, C. (2010). Postembedding labeling methods. Methods Mol Biol 588: 387-395. 
  2. Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17: 208-212. 
  3. Sinetova, M. A., Kupriyanova, E. V., Markelova, A. G., Allakhverdiev, S. I. and Pronina, N. A. (2012). Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii. Biochim Biophys Acta 1817(8): 1248-1255. 

简介

免疫电子显微镜法的目的是将蛋白质定位在莱茵衣藻或其它微藻和蓝细菌的细胞内。 该方案用于研究碳酸酐酶Cah3与在兔中产生的抗体的定位,尽管其可以用于定位任何其他丰富的蛋白质。 建议使用初级兔抗体,因为它们能快速和特异性地与C的蛋白反应。 reinhardtii 。 如果使用除兔以外的第一抗体,应当调整与第一和第二抗体孵育的封闭程序和时间。

关键字:超微结构, 莱茵衣藻, 碳酸酐酶, 定位

材料和试剂

  1. 文化C。 (我们使用来自莫斯科植物生理学研究所微藻类集合的菌株IPPAS D-298)</em> 137mt +
  2. 在兔子中针对感兴趣的蛋白质产生的一抗(我们使用针对莱茵衣藻的重组Cah-3蛋白(α-CA)的抗体,Agrisera,Vannas,Sweden)
  3. 二抗:与10nm胶体金颗粒缀合的抗兔IgG(Sigma-Aldrich,目录号:G7402)
  4. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  5. LR白包埋试剂盒(Sigma-Aldrich,目录号:62662-1EA-F)
  6. 明胶胶囊(SPI供应品,目录号:02308-SS)
  7. Formvar和碳涂层镍网(SPI供应,目录号:3430N-CF)
  8. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A7906)
  9. 山羊血清(Sigma-Aldrich,目录号:G9023)
  10. 兔血清(Sigma-Aldrich,目录号:R4505)
  11. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  12. 乙醇
    可选
  13. 乙酸铀(如 SPI Supplies,目录号:02624-AB)
  14. 柠檬酸铅( Sigma-Aldrich,目录号:15326)
  15. NaOH
  16. 磷酸盐缓冲盐水(PBS)(pH 7.4)(参见配方)
  17. 固定溶液(4%多聚甲醛)(参见配方)
  18. Tris缓冲盐水(TBS)(pH 7.4)(见Recipes)
  19. 1%BSA-TBS(见配方)
  20. 醋酸铀(见配方)
  21. 柠檬酸铅(参见配方)

设备

  1. 96孔免疫板(例如Greiner Bio-One,目录号:650001)。
  2. 带细尖的巴斯德移液器(ROTHE 306.1)
  3. 至少具有37-55°C温度范围的温控器( BINDER B28)
  4. 超薄切片机(例如 Reichert,OMU-3,奥地利)
  5. 网格存储箱(Sigma-Aldrich,目录号:G6276)
  6. 透射电子显微镜(例如,Libra-120,Carl Zeiss,Germany)
  7. 塑料培养皿或具有疏水表面的聚四氟乙烯板
  8. 湿室

程序

  1. 样品制备:
    旋转 C的样本。 在2ml Eppendorf管中在室温下以13,000×g离心5分钟。 所获得的丸粒的最佳体积为足以包埋在2-3个明胶胶囊中的大约50-75μl。
  2. 固定:
    除去上清液,加入0.5ml固定液(4%多聚甲醛)。 为了正确固定,在4℃下的最小培养时间为3小时。
    注意:固定的时间可以延长达数天。 这种固定溶液保存膜和大多数蛋白质,但它可能在几周的储存后脱离淀粉和蛋白质。
  3. 嵌入(所有步骤在室温下进行):
    1. 在相同的Eppendorf管中用PBS缓冲液洗涤沉淀:
      用移液管除去上清液。
      加入1ml PBS缓冲液,轻轻涡旋 孵育5分钟。
      按照步骤1中的说明进行操作。
      重复该洗涤步骤3次。
    2. 脱水颗粒用乙醇稀释系列,每种乙醇浓度两次:
      20%(v/v)15分钟+ 15分钟
      40%(v/v)15分钟+ 15分钟
      50%(v/v)20分钟+ 20分钟
      70%(v/v)20分钟+ 20分钟
      对于每个脱水步骤,丢弃先前的溶液,并用干净的移液管加入0.5ml的下一个顺序溶液。孵育指定的时间。我们建议使用在4°C预冷的新鲜制备的溶液。在这些步骤中小心不要接触或干扰沉淀。
      注意:我 如果团块受到干扰,按照步骤1所述离心Eppendorf管。如果在此步骤有必要中断程序,样品可以在4℃下在70%乙醇中保存过夜。
    3. 渗透:用70%乙醇和LR白色树脂的混合物依次代替70%乙醇:
      3:1 70%乙醇:LR白色1小时
      1:1 70%乙醇:LR白色1小时
      1:3 70%乙醇:LR白色1小时
      接下来的步骤需要用LR White树脂完全代替乙醇:
      用LR White树脂单独代替1:3乙醇/树脂混合物1小时 用新鲜的LR White树脂再次更换,并留置过夜。
      第二天用LR White树脂的新部分替换它,并保留用于嵌入 注意:LR White树脂应根据制造商的方案在使用前与催化剂混合24小时。随着时间的推移,树脂 - 乙醇混合物可以分层成层。如果发生这种情况,用移液管轻轻混合几次(注意不要损伤沉淀)。
    4. 嵌入:
      将明胶胶囊放入96孔免疫板的孔中 在每个明胶胶囊中加入一滴树脂。
      将一小片颗粒放入胶囊中 - 一颗颗粒通常进入2-3粒 用树脂填充胶囊。避免溢出。小心地用盖子盖住胶囊。
      在37℃孵育1-4天(或在45℃孵育一天),然后在55℃孵育直至聚合完成(通常1-3天)。
      注意:在较低温度下较长的温育有助于接受较软的块,较高温度下较短的温育有助于接受更坚固的块,但更脆弱。可以用针检查聚合的完全性: 树脂应该是固体但不易碎。
    5. 用切片机将样品切成切片,并将其放在Formvar和碳涂覆的镍网上 注意:Formvar +碳涂层可防止非特异性标记,并提高切片的质量(无碳层部分经常起皱)。
  4. 免疫细胞化学
    用疏水表面(例如,塑料培养皿或特氟隆板)上的50-100μl液滴的表面上的网格进行所有以下操作。
    提示:为了使网格表面与溶液更好地相互作用,将疏水表面上的液滴和网格放在由于涡旋振动轻微颤抖的桌子上。调整振动的强度,以便液滴表面的颤抖将是明显的。
    当需要过夜培养栅格时,将其放入4℃的湿室中。在后续步骤中不允许网格变干。
    1. 阻断抗体在BSA-TBS中的20%山羊血清的滴的非特异性相互作用1小时 注意:如果仍然发生非特异性标记,请将山羊血清高达50%和BSA高达5%的比例增加。
    2. 将网格放在100微升滴与适当稀释的一抗针对目标蛋白质。稀释度可以仅凭经验确定;通常在1:16 - 1:128的范围内。
      对于阴性对照,将兔血清或更好地,预免疫血清而不是一抗施用于靶蛋白 将纯化的目标蛋白加入作为阳性对照的一抗的溶液中 离开网格过夜在4℃。您可以改变时间和温度以增加或减少结合的效率和标签的强度
    3. 用TBS洗涤滴液上的网格5次,在TBS溶液的更换之间保持滴液5-10分钟
    4. 将网格转移到100微升适当稀释(根据制造商的协议)的抗兔IgG与BSA-TBS中的10nm胶体金颗粒缀合并在室温下暴露1小时。
    5. 用TBS洗涤网格,用TBS洗涤5次,然后用去离子水洗涤。这些洗涤步骤应通过在1分钟内快速重复地将网格浸入3-5ml TBS和水中来进行。
    6. 干滤网放在滤纸上,放入网格储存箱
    7. 可选 - 依次将网格与乙酸铀酰和柠檬酸铅对比。用NaOH晶体和用0.2M NaOH润湿的滤纸制备湿室(图1)。将网格放在100微升醋酸双氧铀滴45-60分钟。倒入50μl的柠檬酸铅到同一板。通过快速反复浸没到3-5ml的0.2M NaOH(5-10次),然后 - 3-5毫升煮沸的去离子水(1分钟)洗涤网格。需要NaOH溶液和沸水以避免CO 2与铅的反应,这导致不溶性晶体的形成。将网格放在制备的柠檬酸铅滴5-7分钟。以与在乙酸铀酰之后进行的相同的方式洗涤。干滤网放在滤纸上,放入网格储存箱中。
  5. 用电子透射显微镜分析切片 注意:在阴性和阳性对照中,只允许有少量金颗粒作为背景。


    图1.湿室用NaOH晶体进行对比染色

食谱

  1. 磷酸盐缓冲盐水(PBS)(pH 7.4)
    1. 准备储备溶液:
      储液1:NaH 2 PO 4 4.76g/100ml
      储备液2:Na 2 HPO 4 14.2g/500ml
    2. 对于0.5L PBS缓冲液,混合95ml的储备液1和405ml的储液2,并加入
      4.25g NaCl(终浓度为0.85%) 此溶液的pH应为7.4(可用NaOH调节)
  2. 固定溶液(4%多聚甲醛)
    1. 将50ml PBS(pH7.4)加热至70℃
    2. 在热PBS中搅拌溶解2g多聚甲醛
    3. 在室温下冷却溶液,并通过滤纸
      过滤 (保持溶液在4°C在黑暗中,使用1周内)
  3. Tris缓冲盐水(TBS)(pH7.4) Tris-HCl 2.4g/L
    NaCl 8.76g/L
    用HCl
    调节pH至7.4
  4. 1%BSA-TBS
    向TBS中加入10 g/L的BSA
    保持等分试样冷冻在-20°C,或使用新鲜。
  5. 乙酸乙烯酯
    制备乙酸铀酰在50%乙醇中的饱和溶液。 通过滤纸过滤。 使用新鲜。
  6. 柠檬酸铅
    1. 将1.33g的Pb(NO 3)2和1.76g的Na 3(C 6 H 5) 在30ml去离子水中的溶液中,加入10%氢氧化钠水溶液。 在轨道振荡器中摇动30分钟。 在这个阶段,解决方案可能显得乳白色
    2. 加入8ml 1M NaOH:溶液应变得透明。调整 用去离子水将体积调节至50ml。 pH应该约为12.
      保持小等分在-20°C。使用前解冻和旋转。

致谢

该方案改编自Sinetova等人(2012)。我们赞赏J.Moroney教授(美国路易斯安那州立大学)提供C的应变。我们为我们提供了针对重组Cah3和Cah3-MBP融合蛋白的一级抗体。我们感谢Pronina N.A.教授和Kupriyanova E.V.博士为本方案的生理和分子生物学基础。这项工作是由俄罗斯基础研究基金会(第10-04-01463号),俄罗斯科学院院长授予的资金(方案"分子和细胞生物学")和俄罗斯的拨款教育和科学部(16.740.11.0176)。

参考文献

  1. Oliver,C。(2010)。 嵌入标签方法。 Methods Mol Biol 588:387-395。 
  2. Reynolds,E.S。(1963)。 在高pH下使用柠檬酸铅作为电子显微镜中的电子不透明染料。 J Cell Biol 17:208-212。 
  3. Sinetova,M.A.,Kupriyanova,E.V.,Markelova,A.G.,Allakhverdiev,S.I.and Pronina,N.A。(2012)。 碳酸酐酶Cah3在灵芝衣藻类囊体膜中的鉴定和功能作用/em> 。 Biochim Biophys Acta 1817(8):1248-1255。 
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sinetova, M. A. and Markelova, A. G. (2013). Immunoelectron Microscopy in Chlamydomonas Cells. Bio-protocol 3(5): e335. DOI: 10.21769/BioProtoc.335.
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