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A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells
植物细胞中高尔基体和其它胞器的膜转运调节因子的生物成像

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Abstract

The plant Golgi apparatus is composed of numerous stacks of cisterna, designated as cis, medial, and trans Golgi cisternae; these stacks move within the cytoplasm along the actin cytoskeleton. Cis cisternae receive secretory products from endoplasmic reticulum (ER) and they subsequently progress through the stack to the trans cisternae, where they are sorted to other destinations, including cell wall, plasma membrane (PM), vacuoles, and chloroplasts. In addition, the plant Golgi apparatus plays a role of glycosylating proteins as well as synthesizing cell wall polysaccharides, such as hemicelluloses and pectins. This protocol describes procedures for imaging fluorescently-tagged proteins localized to the plant Golgi apparatus of Arabidopsis seedlings using confocal laser microscopy (CLSM), total internal reflection fluorescence microscope (TIRF), and immunogold labeling of high-pressure frozen/freeze substituted samples by transmission electron microscopy (TEM). We particularly focus on long-term time lapse imaging and protein localization in subdomains within the Golgi. This protocol can be also used for other organelles, tissues, and plant species.

Materials and Reagents

  1. ½X Murashige and Skoog (MS) medium (Sigma-Aldrich, catalog number: M5519 )
  2. Bacto Agar [Becton, Dickinson and Company (BD), catalog number: 2140101 ]
  3. Arabidopsis thaliana seedlings, expressing Golgi-localized proteins fused to a fluorescent protein (e.g. 35S::ST-mRFP in Col, 35S::ERD2-GFP in Col, pGNL1:: GNL1-YFP in gnl1, pGNOM::GNOM-GFP in gnom)
  4. Inhibitors [such as BrefeldinA (BFA) (Sigma-Aldrich, catalog number: B7651 ), monensin (Sigma-Aldrich, catalog number: M5273 ), salinomycin (Sigma-Aldrich, catalog number: S4526 )]
  5. Dimethyl sulfoxide (DMSO) (Wako Chemicals USA, catalog number: 046-2198 )
  6. Freezing planchets type B (Ted Pella, catalog number: 39201 )
  7. Sucrose (Wako Chemicals USA, catalog number: 196-00015 )
  8. 1.8 ml cryovials [for example: Nunc® cryotubes (Sigma-Aldrich, catalog number: V7884-450EA )]
  9. Methacrylate-based resin (Lowicryl HM20 resin kit) (Electron Microscopy Sciences, catalog number: 14340 )
  10. Sodium phosphate monobasic (NaH2PO4) (Sigma-Aldrich, catalog number: S8282-500G )
  11. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S7907-100G )
  12. Liquid nitrogen
  13. 0.5% Formvar solution (Electron Microscopy Sciences, catalog number: 15820 )
  14. Tween-20 (Sigma-Aldrich, catalog number: P1379-25ML )
  15. Primary antibodies against fluorescent tag [e.g. green fluorescent proteins(GFP)]
  16. Secondary antibodies conjugated to gold nanoparticles (5, 10, or 15 nm in diameter)
  17. Cryo-substitution solution (see Recipes)
  18. Lead citrate (see Recipes)
  19. Uranyl actetate solution (see Recipes)
  20. Phosphate-buffer saline (PBS) stock solution (10x) (see Recipes)
  21. 0.1% Tween-20 in PBS (PBS-T-0.1%) (see Recipes)
  22. 0.5% Tween-20 in PBS (PBS-T-0.5%) (see Recipes)
  23. Blocking buffer (see Recipes)
  24. HM20 resin solutions (see Recipes)

Equipment

  1. Coverwell® silicone imaging chambers (2.5 mm deep, 20 mm diameter) (Electron Microscopy Sciences, catalog number: 70326-16 )
  2. Nickel slot grids (Electron Microscopy Sciences, catalog number: 2015-Ni )
  3. 0.12-0.17 mm thick coverslips (Matsunami Glass, catalog number: C022221 )
  4. Light Forceps (Hammacher, catalog number: HWC 118-10 )
  5. Lab-Tek Chambered Coverglass (Thermo Fisher Scientific, catalog number: 155361 )
  6. 0.9-1.2 mm thick Glass slides (Matsunami Glass, catalog number: S24410 )
  7. Parafilm
  8. 22 °C plant growing chamber
  9. Inverted confocal Microscope (Olympus, model: FV1200 )
    Note: equipped with 473 nm or 559 nm diode laser and with water immersion 63x / 1.20 numerical aperture objectives or oil immersion 100x / 1.40 numerical aperture objectives
  10. TIRF Microscope equipped with oil immersion CFI Apo TIRF 100x H / 1.49 numerical aperture objectives (Nikon, model: Eclipse TE2000-E with TIRF2 system)
  11. High-pressure freezer (Leica, model: EM HPM100 or Bal-tec/RMC/ABRA Fluid AG, model: HPM 010 )
  12. Automated freeze-substitution and low-temperature resin embedding/polymerization system (for example, Leica, model: AFS2 )
  13. Ultramicrotome (for example, Leica, model: EM UC7 ultramicrotome )
  14. Glass knife maker (for example, Leica, model: EM KMR3 )
  15. Ultra 45° diamond knife or other types of wet diamond knives (Diatome AG)
  16. Transmission electron microscope (for example, FEI, model: Tecnai 12 )

Procedure

  1. Live imaging of Golgi apparatus by using Confocal microscope
    This protocol is suitable for performing the long-term live imaging of plant cells.
    1. Put seeds on ½X MS media solidified with 0.8% Bacto agar and grow them vertically at 22 °C for 4-5 days under continuous light.
    2. Collect 4-5 days old Arabidopsis seedlings and transfer them onto ½X MS media solidified with 0.8% Bacto agar, including inhibitor or control DMSO solvent.
      Note: To prevent damaging root cells of Arabidopsis seedlings, transferring Arabidopsis seedlings by holding cotyledons with light forceps are recommended. Inhibitor stocks are dissolved in DMSO and thus corresponding amount of DMSO should be contained in control medium. Working concentration of inhibitors can vary depending on chemicals and cell types analyzed but it is normally 10-100 µM.


      Figure 1. Preparing specimen for long-term live cell imaging using Arabidopsis seedlings. A. Required materials. B-I. Sample preparation for imaging Arabidopsis cells.

    3. Transfer Arabidopsis seedlings and solid media onto the Lab-Tek Chambered Coverglass. Seedlings should be sandwiched between coverslips and solid media (see Video 1 and Figure 1).

      Video 1. Specimen preparation for long-term live cell imaging using Arabidopsis seedlings

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    4. Place prepared Lab-Tek Chambered Coverglass on the inverted confocal laser microscope equipped with 63x or 100x objectives.
    5. Focus specimen and observe cells.
      Note: Observing cells located close to the coverslip, such as epidermal cells, can provide better image. For performing long-term time lapse imaging, be sure that the objective does not dry out. To prevent water immersion objective from drying, place one drop of Zeiss Immersol W directly onto the objectives or coverslips as immersion medium. As a marker of Golgi apparatus, fluorescently labeled ERD2, ST, COPI proteins can be used (Naramoto et al., 2014). Please note that this experimental setup allows for limited gas exchange, which might affect several processes in the plant. Alternatives for long-term imaging include imaging chambers or microfluidic devices for plants (Busch et al., 2012).

  2. Detailed live-imaging analysis of Golgi-localized proteins by using TIRF microscopy
    This protocol is suitable for visualizing the detailed subcellular localization of proteins that localized around cell surface.
    1. Place seeds on ½X MS solid media (0.8% Bacto agar) and grow them vertically on 22 °C for 7 days under the continuous light.
    2. Transfer 7-day-old Arabidopsis seedlings into ½X MS liquid medium and incubate them for longer than 30 min. If necessary, inhibitor or DMSO control solvent can be added.
      Note: Incubation time can vary depending on the experiments but it is normally from 30 min to 2 h.
    3. Excise root or hypocotyl segments from 7-day-old Arabidopsis seedlings by using fine scissors or surgical knives (see Figure 2).


      Figure 2. Excision of Arabidopsis roots for TIRF microscopy imaging. A. 7-day-old Arabidopsis seedlings. B. 7-day-old Arabidopsis seedlings, excised by scissor. Excision position at roots or hypocotyls are indicated by dashed white line.

    4. Mount root or hypocotyl segments with liquid media using glass slides and cover slips.
    5. Place prepared specimen on the TIRF microscope equipped with an oil immersion CFI Apo TIRF 100x H/1.49 numerical aperture objective.
    6. Focus the specimen and observe cells.
      Note: Only cells in contact with the surface of coverslip can be imaged. For the roots, cells in elongation zone, especially those close to the differentiation zone, are suitable for observation. Please see example TIRF image of pGNOM::GNOM-GFP that localize at Golgi apparatus (see Video 2).

      Video 2. Live imaging of GNOM-GFP localized at Golgi apparatus by TIRF microscopy as described in this protocol

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  3. Immunogold labeling of Golgi localized proteins by using TEM
    1. Submerge seedlings grown on ½ MS plates and expressing a fluorescently-tagged Golgi-localized protein in a drop of 0.1 M sucrose. Cut 1 mm-long root segments and place them inside freezing planchet (Figure 3A) filled with 0.1 M sucrose. Besides transgenic roots expressing fluorescently-tagged proteins, process also wild-type samples to use as negative controls during immunolabeling.


      Figure 3. Preparation of root samples for immunogold labeling. A. Freezing planchets for high-pressure freezing. B. Mounting of HM20 resin-embedded roots on stubs for sectioning.

    2. Place another freezer planchet on top, flat side down, to close the chamber.
    3. Freeze the two planchets containing the root segments in a high-pressure freezer.
    4. Under liquid nitrogen, separate the two planchets and transfer the one containing the frozen roots to a cryovial with cryosubstitution solution.
    5. Place cryovial with cryosubstitution solution and samples into a cryosubstitution device pre-cooled to -90 °C. Samples should remain at -90 °C for at least 3 days.
    6. Raise the temperature of the cryosubstitution device to -60 °C. Remove cryosubstitution solution and rinse samples with fresh, pre-cooled (-60 °C) anhydrous acetone 3-4 times. Remove empty freezing planchets using pre-cooled tweezers (all root segments should be detached from the planchets and free inside in the cryovial with acetone).
    7. Remove acetone and add pre-cooled (-60 °C) 30% HM20 solution for at least 3 h. Repeat this step with pre-cooled (-60 °C) 60% HM20 solution (3 h) and 100% HM20 (3 h). At least 3 more changes with 100% HM20 are recommended. During the entire embedding procedures, samples should remained at -60 °C.
    8. Assemble embedding chambers by attaching Coverwell® imaging chambers to a glass slide. Fill embedding chambers with fresh 100% HM20 and transfer roots from cryovial to embedding chambers. Cover with a glass coverslip.
    9. Polymerize HM20 resin at -50 °C with UW light for 48 h.
    10. Once polymerized, remove resin blocks from embedding chambers. Locate roots within resin block with the help of a dissecting microscope. Cut pieces or resin containing roots and mount them on top of resin stubs using super glue (Figure 3B).
    11. With a razor blade trim the resin block around the root. Start sectioning with a glass knife in an ultramicrotome until reaching the root. Switch to a diamond knife; collect 70 nm-thick sections on Formvar coated-nickel grids.


      Figure 4. Immunogold labeling of GNOM-GFP with anti-GFP antibodies on high-pressure frozen-freeze substituted Arabidopsis roots. A. Detection of GNOM-GFP in transgenic roots treated for 3 min in DMSO. B. Detection of GNOM-GFP in transgenic roots treated for 3 min in BFA. C. Negative control: wild type roots treated with DMSO for 3 min. Arrowheads indicate gold particles. G, Golgi; MVB, multivesicular body. TGN, Trans Golgi Network. Scale bars = 200 nm. Modified from Naramoto et al. (2014) Copyright American Society of Plant Biologists.

    12. Place 10 μl drops of blocking buffer on a piece of parafilm and float nickel grids on top of drops (the root sections should be in contact with the blocking buffer) for 15-20 min.
    13. Transfer grids to 10 μl drops of primary antibody diluted in blocking buffer (recommended dilutions: 1:10 to 1:50) for 1 h.
    14. Rinse grids under a stream of PBS-T-0.5% buffer for 1 min. Blot grids with filter paper and float them on 10 μl drops of secondary antibody solution (recommended dilutions 1:10 to 1:100) for 1 h.
    15. Rinse grids with PBS-T-0.5% buffer for 1 min followed by a rinse with distilled water.
    16. Stain root sections with uranyl acetate solution for 10 min followed by lead citrate for 5 min.
    17. Observed sections in a transmission electron microscope (Figure 4).

Recipes

  1. Cryo-substitution solution
    0.2% glutaraldehyde plus 0.2% uranyl acetate in acetone
    Place 1.5 ml in a 2 ml cryovial and store individual aliquots in liquid nitrogen
  2. Lead citrate
    1. Wear gloves to handle lead nitrate
      Boil 50 ml of distilled water to remove CO2 (CO2 dissolved in water can cause this solution to precipitate) and let the water cool down at room temperature
    2. Add 0.33 g of lead nitrate to 10 ml of boiled distilled water and mix gently until the lead nitrate crystals completely dissolve
    3. Add 0.44 g of sodium citrate and mixed gently; the solution will become milky white
    4. Add 2 ml of 1 N sodium hydroxide solution freshly prepared with boiled water; the solution will become transparent
    5. Bring up volume to 12.5 ml with boiled distilled water
    6. Store solution at either room temperature or 4 °C
  3. Uranyl actetate solution
    2% uranyl acetate (w/v) in 30% methanol (v/v)
  4. Phosphate-buffer saline (PBS) stock solution (10x)
    1.76 g of NaH2PO4, 11.49 g of Na2HPO4, 85 g sodium chloride in 1 L of distilled water, pH 6.8 (store at room temperature)
  5. 0.1% Tween-20 in PBS (PBS-T-0.1%)
    Add 100 μl of Tween-20 to 100 ml of 1x PBS
    Wash Tween-20 out of the pipette tip by pipetting up and down several times into the PBS-T-0.1% solution
  6. 0.5% Tween-20 in PBS (PBS-T-0.5%)
    Add 5 ml of Tween-20 to 1 L of 1x PBS
  7. Blocking buffer
    10% (w/v) nonfat dry milk in PBS-T-0.1%
    For a 10 ml volume, add PBS-T-0.1% to 1 g of nonfat dry milk up to 10 ml final volume
  8. HM20 resin solutions
    Prepare HM20 resin in the hood according to manufacturer’s instructions
    Make 30% and 60% HM20 resin solutions (v/v) by mixing with the appropriate volume of anhydrous acetone

Acknowledgements

This work was supported by Grant for Basic Science Research Projects from The Sumitomo Foundation to SN; the Japanese Society for the Promotion of Science (JSPS; 30612022 to S.N.); the Ministry of Education, Culture, Sports, Science and Technology in Japan [NC-CARP project (to S.N.)] and U. S. National Science Foundation grant MCB1157824 (to MSO).

References

  1. Busch, W., Moore, B. T., Martsberger, B., Mace, D. L., Twigg, R. W., Jung, J., Pruteanu-Malinici, I., Kennedy, S. J., Fricke, G. K., Clark, R. L., Ohler, U. and Benfey, P. N. (2012). A microfluidic device and computational platform for high-throughput live imaging of gene expression. Nat Methods 9(11): 1101-1106.
  2. Naramoto, S., Otegui, M. S., Kutsuna, N., de Rycke, R., Dainobu, T., Karampelias, M., Fujimoto, M., Feraru, E., Miki, D., Fukuda, H., Nakano, A. and Friml, J. (2014). Insights into the localization and function of the membrane trafficking regulator GNOM ARF-GEF at the Golgi apparatus in Arabidopsis. Plant Cell 26(7): 3062-3076.

简介

植物高尔基体包括许多叠状体,称为顺,内侧和反式高尔基这些堆栈在细胞质内沿着肌动蛋白细胞骨架移动。 Ctern 池从内质网(ER)接收分泌产物,并且随后通过堆叠进入反转池,在那里它们被分选到其他目的地,包括细胞壁,血浆膜(PM),液泡和叶绿体。此外,植物高尔基体起到糖基化蛋白质以及合成细胞壁多糖如半纤维素和果胶的作用。该协议描述了使用共聚焦激光显微镜(CLSM),全内反射荧光显微镜(TIRF)和高压的免疫金标记标记定位于拟南芥幼苗的植物高尔基体的荧光标记的蛋白质的程序冷冻/冷冻取代的样品通过透射电子显微镜(TEM)。我们特别关注长期时间推移成像和蛋白质本地化在高尔基内的子域。该方案还可以用于其他细胞器,组织和植物物种。

材料和试剂

  1. 1/2X Murashige和Skoog(MS)培养基(Sigma-Aldrich,目录号:M5519)
  2. Bacto Agar [Becton,Dickinson and Company(BD),目录号:2140101]
  3. 拟南芥(Arabidopsis thaliana)苗,其表达与荧光蛋白融合的高尔基体定位的蛋白质(例如 35S :: ST-mRFP :: ERD2-GFP in col, pGNL1 :: GNL1-YFP in gnl1 , pGNOM :: GNOM-GFP in gnom )
  4. 抑制剂[诸如BrefeldinA(BFA)(Sigma-Aldrich,目录号:B7651),莫能菌素(Sigma-Aldrich,目录号:M5273),沙利霉素(Sigma-Aldrich,目录号:S4526)
  5. 二甲基亚砜(DMSO)(Wako Chemicals USA,目录号:046-2198)
  6. 冷冻型B型(Ted Pella,目录号:39201)
  7. 蔗糖(Wako Chemicals USA,目录号:196-00015)
  8. 1.8ml冷冻管[例如:Nunc cryotubes(Sigma-Aldrich,目录号:V7884-450EA)]
  9. 甲基丙烯酸酯类树脂(Lowicryl HM20树脂试剂盒)(Electron Microscopy Sciences,目录号:14340)
  10. 磷酸二氢钠(NaH 2 PO 4)(Sigma-Aldrich,目录号:S8282-500G)
  11. 磷酸氢二钠(Na 2 HPO 4)(Sigma-Aldrich,目录号:S7907-100G)
  12. 液氮
  13. 0.5%Formvar溶液(Electron Microscopy Sciences,目录号:15820)
  14. Tween-20(Sigma-Aldrich,目录号:P1379-25ML)
  15. 针对荧光标签的初级抗体[例如绿色荧光蛋白(GFP)]
  16. 与金纳米颗粒(直径5,10或15nm)结合的二抗
  17. 低温替代溶液(参见配方)
  18. 柠檬酸铅(参见配方)
  19. 铀酰乙酸溶液(参见配方)
  20. 磷酸盐缓冲盐水(PBS)储备溶液(10x)(见配方)
  21. 0.1%Tween-20的PBS溶液(PBS-T-0.1%)(参见Recipes)
  22. 0.5%Tween-20的PBS(PBS-T-0.5%)(参见Recipes)
  23. 阻止缓冲区(参见配方)
  24. HM20树脂溶液(见配方)
    柠檬酸铅(参见配方)
  25. 铀酰乙酸溶液(参见配方)
  26. 磷酸盐缓冲盐水(PBS)储备溶液(10x)(见配方)
  27. 0.1%Tween-20的PBS溶液(PBS-T-0.1%)(参见Recipes)
  28. 0.5%Tween-20的PBS(PBS-T-0.5%)(参见Recipes)
  29. 阻止缓冲区(参见配方)
  30. HM20树脂溶液(见配方)
    ... 柠檬酸铅(参见配方)
  31. 铀酰乙酸溶液(参见配方)
  32. 磷酸盐缓冲盐水(PBS)储备溶液(10x)(见配方)
  33. 0.1%Tween-20的PBS溶液(PBS-T-0.1%)(参见Recipes)
  34. 0.5%Tween-20的PBS(PBS-T-0.5%)(参见Recipes)
  35. 阻止缓冲区(参见配方)
  36. HM20树脂溶液(见配方)
    ...... 柠檬酸铅(参见配方)
  37. 铀酰乙酸溶液(参见配方)
  38. 磷酸盐缓冲盐水(PBS)储备溶液(10x)(见配方)
  39. 0.1%Tween-20的PBS溶液(PBS-T-0.1%)(参见Recipes)
  40. 0.5%Tween-20的PBS(PBS-T-0.5%)(参见Recipes)
  41. 阻止缓冲区(参见配方)
  42. HM20树脂溶液(见配方)
    ......... 程序

    1. 使用共聚焦显微镜实时成像高尔基体 此协议适合于执行植物细胞的长期实时成像。
      1. 将种子放在1/2X MS培养基上,用0.8%Bacto琼脂固化,并在22℃下在连续光照下垂直生长4-5天。
      2. 收集4-5天的拟南芥幼苗并将其转移到1/2X   MS培养基用0.8%Bacto琼脂固化,包括抑制剂或 控制DMSO溶剂 注意:防止损伤根细胞 拟南芥幼苗,通过保持转移拟南芥幼苗 推荐使用轻质镊子的子叶。 抑制剂库存 溶于DMSO中,因此相应量的DMSO应该是 包含在对照培养基中。 抑制剂的工作浓度 根据所分析的化学品和细胞类型而变化,但通常是这样 10-100μM。


        图1.使用拟南芥幼苗制备用于长期活细胞成像的标本。 A.所需材料。双。样品制备 用于成像拟南芥细胞。

      3. 将拟南芥幼苗和固体培养基转移到 Lab-Tek Chambered Coverglass。幼苗应夹在中间  盖玻片和固体介质(见视频1和图1)
        视频1.使用拟南芥幼苗进行长期活细胞成像的标本准备
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      4. 将准备好的Lab-Tek Chambered盖玻片放在装有63x或100x物镜的倒置共聚焦激光显微镜上
      5. 聚焦标本并观察细胞 注意:观察靠近盖玻片的细胞,例如表皮   细胞,可以提供更好的图像。 用于执行长期时间流逝 成像,确保目标不干透。 防止水 浸渍物镜干燥,放置一滴Zeiss Immersol W 直接在目标或盖玻片上作为浸没介质。 作为一个 高尔基体标记,荧光标记的ERD2,ST,COPI蛋白   可以使用(Naramoto等,2014)。 请注意这个实验 设置允许有限的气体交换,这可能影响几个 过程在植物中。 长期成像的替代方法包括 成像室或植物的微流体装置(Busch等人, 2012)。

    2. 使用TIRF显微镜进行高尔基体定位的蛋白质的详细的活体成像分析 该协议适合于可视化细胞表面周围的蛋白质的细胞亚细胞定位
      1. 将种子放在1/2MS固体培养基(0.8%Bacto琼脂)上,并在连续光下在22℃下垂直生长7天。
      2. 将7日龄拟南芥幼苗转移到1X MS液体培养基中 并孵育超过30分钟。 如有必要,抑制剂或 可以加入DMSO对照溶剂。
        注意:孵育时间可能因实验而异,但通常为30分钟至2小时。
      3. 通过使用细剪刀或手术刀(见图2)从7日龄拟南芥苗培育根或下胚轴段。


        图2.用于TIRF显微镜成像的拟南芥根的切除。 7天的拟南芥幼苗。 B.7天大的拟南芥幼苗, 由剪刀切除。 在根或下胚轴处的切除位置是 用白色虚线表示。

      4. 使用玻璃载玻片和盖玻片用液体培养基安装根部或下胚轴段。
      5. 将准备好的样品放在装有油的TIRF显微镜上 浸没式CFI Apo TIRF 100x H/1.49数值孔径物镜
      6. 聚焦样本并观察细胞 注意:只有与盖玻片表面接触的细胞可以 成像。对于根,细胞在伸长区,特别是那些靠近 到分化区,适合观察。请参见 例如定位于高尔基体的pGNOM :: GNOM-GFP的TIRF图像 (见视频2)。

        视频2.通过本协议中描述的TIRF显微镜定位于高尔基体的GNOM-GFP的活体成像
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    3. 通过使用TEM免疫金标记高尔基体本地化的蛋白质
      1. 将秧苗在1/2MS板上生长并表达a 荧光标记的高尔基定位的蛋白质在一滴0.1M蔗糖中。   切割1毫米长的根段,并将其放置在冷冻棘轮内 (图3A)。 除转基因根 表达荧光标记蛋白,也处理野生型样品   在免疫标记期间用作阴性对照。


        图3。 制备用于免疫金标记的根样品。 A.冷冻 高压冷冻平台。 B.安装HM20树脂嵌入 根在桩上切片。

      2. 将另一个冰柜平面顶部,平面朝下放置,关闭室。
      3. 在高压冰箱中冻结包含根段的两个平面
      4. 在液氮下,分开两个平板并转移 一个含有冷冻根到具有冷冻取代的冷冻管 解决方案
      5. 放置冷冻管与冷冻补液溶液和 样品置于预冷却至-90℃的冷冻置换装置中。 样品 应保持在-90°C至少3天。
      6. 提高 冷却装置的温度为-60℃。 去掉 冷冻取代溶液,并用新鲜,预冷却(〜60℃)冲洗样品 ℃)无水丙酮3-4次。 使用删除空冷冻平面 预冷镊子(所有根段应从中分离 平面和自由内部在冷冻管与丙酮)。
      7. 去掉 丙酮并加入预冷(-60℃)30%HM20溶液至少3小时。 用预冷却(-60℃)的60%HM20溶液(3小时)和重复该步骤 100%HM20(3h)。 建议使用100%HM20进行至少3次更改。   在整个嵌入过程中,样品应保持在-60 ℃。
      8. 通过连接Coverwell ®成像组装嵌入室 室到玻璃载玻片。 填充与新鲜100%HM20的嵌入室 并将根从冷冻管转移到包埋室。 封面用 玻璃盖玻片
      9. 在-50℃下用UW光聚合HM20树脂48小时
      10. 一旦聚合,从嵌入室去除树脂块。 在解剖的帮助下找到树脂块内的根 显微镜。 切割块或树脂含根,并将其安装在顶部 的树脂桩使用超级胶水(图3B)
      11. 用剃刀刀片 修剪树根块周围的树脂块。 开始用玻璃切片 刀在超薄切片机中直到到达根部。 切换到钻石 刀; 在Formvar涂层镍网上收集70nm厚的部分

        图4.用抗GFP抗体对GNOM-GFP进行免疫金标记 高压冷冻取代的拟南芥根。 A.检测 的GNOM-GFP在DMSO中处理3分钟的转基因根。 B.检测 的GNOM-GFP在BFA中处理3分钟的转基因根。 C.负面 对照:用DMSO处理3分钟的野生型根。 箭头 表示金颗粒。 G,Golgi; MVB,多泡体。 TGN,Trans 高尔基网络。 比例尺= 200nm。 修改自Naramoto等人(2014)   版权所有美国植物生物学家协会
      12. 地方10 μl的封闭缓冲液在一块石蜡膜和浮法镍上 网格在滴的顶部(根部应该与 封闭缓冲液)15-20分钟。
      13. 转移网格到10微升的一抗在封闭缓冲液(推荐的稀释度:1:10到1:50)稀释1小时。
      14. 在PBS-T-0.5%缓冲液流下漂洗网格1分钟。 污点 网格用滤纸和浮动他们在10微升次要 抗体溶液(建议稀释度1:10至1:100)1小时。
      15. 用PBS-T-0.5%缓冲液冲洗网格1分钟,然后用蒸馏水冲洗
      16. 用乙酸铀酰溶液染色根切片10分钟,然后用柠檬酸铅染色5分钟。
      17. 在透射电子显微镜中观察切片(图4)。

    食谱

    1. 低温替代溶液
      0.2%戊二醛加0.2%乙酸双氧铀在丙酮中的溶液 将1.5 ml置于2ml冷冻管中,并将单个等分试样储存在液氮中
    2. 柠檬酸铅
      1. 戴手套处理硝酸铅
        煮沸50ml蒸馏水以除去CO 2(CO 2溶解于水中可导致该溶液沉淀),并让水在室温下冷却
      2. 向10ml蒸馏水中加入0.33g硝酸铅,轻轻混合直至硝酸铅晶体完全溶解
      3. 加入0.44克柠檬酸钠,轻轻混匀; 溶液将变成乳白色
      4. 加入2ml用煮沸水新鲜制备的1N氢氧化钠溶液; 该解决方案将变得透明
      5. 用蒸馏的蒸馏水将体积调至12.5ml
      6. 将溶液储存在室温或4℃下
    3. 乙酸铀酰溶液
      2%乙酸双氧铀(w/v)在30%甲醇(v/v)中的溶液
    4. 磷酸盐缓冲盐水(PBS)储备溶液(10x)
      将1.76g的NaH 2 PO 4,11.49g的Na 2 HPO 4,85g的氯化钠溶于1ml二氯甲烷中, L的蒸馏水,pH6.8(在室温下保存)
    5. 0.1%Tween-20的PBS溶液(PBS-T-0.1%) 将100μlTween-20加入100 ml 1x PBS中
      用移液器上下移动几次将Tween-20移出移液管吸头,进入PBS-T-0.1%溶液中
    6. 0.5%Tween-20的PBS溶液(PBS-T-0.5%) 将5毫升Tween-20加到1升1×PBS中
    7. 阻塞缓冲区
      10%(w/v)脱脂奶粉的PBS-T-0.1%
    8. HM20树脂溶液
      根据制造商的说明在护罩中准备HM20树脂
      通过与适当体积的无水丙酮混合,制备30%和60%HM20树脂溶液(v/v)

    致谢

    这项工作是从住友基金会到SN的基础科学研究项目的拨款支持;日本科学促进会(JSPS; 30612022 to S.N.);日本教育,文化,体育,科学和技术部[NC-CARP项目(到S.N.)]和美国国家科学基金会授予MCB1157824(MSO)。

    参考文献

    1. Busch,W.,Moore,BT,Martsberger,B.,Mace,DL,Twigg,RW,Jung,J.,Pruteanu-Malinici,I.,Kennedy,SJ,Fricke,GK,Clark,RL,Ohler,和Benfey,PN(2012)。 用于基因表达的高通量实时成像的微流体装置和计算平台。 > Nat Methods 9(11):1101-1106。
    2. Naramoto,S.,Otegui,MS,Kutsuna,N.,de Rycke,R.,Dainobu,T.,Karampelias,M.,Fujimoto,M.,Feraru,E.,Miki,D.,Fukuda, Nakano,A。和Friml,J。(2014)。 观察膜运输调节剂GNOM ARF-GEF在拟南芥中的高尔基体的定位和功能。 。 Plant Cell 26(7):3062-3076。

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引用:Naramoto, S., Dainobu, T. and Otegui, M. S. (2015). A Bioimaging Pipeline to Show Membrane Trafficking Regulators Localized to the Golgi Apparatus and Other Organelles in Plant Cells. Bio-protocol 5(17): e1583. DOI: 10.21769/BioProtoc.1583.
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