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Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides
拟南芥粘液多糖的全种子免疫标记   

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参见作者原研究论文

本实验方案简略版
Plant Physiology
Dec 2015

Abstract

In addition to synthesizing and secreting copious amounts of pectic polymers (Young et al., 2008), Arabidopsis thaliana seed coat epidermal cells produce small amounts of cellulose and hemicelluloses typical of secondary cell walls (Voiniciuc et al., 2015c). These components are intricately linked and are released as a large mucilage capsule upon hydration of mature seeds. Alterations in the structure of minor mucilage components can have dramatic effects on the architecture of this gelatinous cell wall. The immunolabeling protocol described here makes it possible to visualize the distribution of specific polysaccharides in the seed mucilage capsule.

Keywords: Arabidopsis thaliana (拟南芥), Plant cell wall (植物细胞壁), Immunolabeling (免疫标记), Carbohydrate (糖类), Fluorescence (荧光), Seed coat (种皮), Microscopy (显微镜检查)

Background

Since the first comprehensive immunofluorescence analysis of pectin-rich mucilage in Arabidopsis seed coat epidermal cells (Young et al., 2008), additional types of polysaccharides have been detected in this specialized cell wall (Voiniciuc et al., 2015a; 2015b and 2015c). To handle more samples in parallel, I adapted the original protocols (performed in 1.5-ml microcentrifuge tubes; Young et al., 2008; Harpaz-Saad et al., 2011) to a 24-well plate format. I recommend counterstaining seeds with Pontamine S4B, a fluorescent dye that is more specific to cellulose than previous stains (Anderson et al., 2010). By testing for cross-talk between multiple fluorophores and setting clear guidelines for image acquisition and processing, this protocol yields reproducible mucilage phenotypes that can be reliably interpreted.

Materials and Reagents

  1. Personal protection equipment (safety glasses, lab coat, gloves)
  2. 1.5 ml snap-cap tubes
  3. 24-well plate with lid (VWR, catalog number: 734-2325 )
  4. Plastic Pasteur pipette (VWR , catalog number: LSUK711117S/20 )
  5. Manual pipettes tips (Eppendorf, Research plus and Repeater plus, or similar style)
  6. Aluminum foil
  7. MARIENFELD glass slides, low autofluorescence, L x B x H: 76 x 26 x 1 mm; pre-washed; Ground edges, 90° slides (VWR, catalog number: 631-9464 )
  8. Precision cover slip glass, thickness No. 1.5H (Marienfeld-Superior, catalog number: 0107222 )
  9. 15 ml Falcon tubes (VWR, catalog number: 734-0452 )
  10. Arabidopsis thaliana mature, dry seeds
  11. Primary antibodies directed against plant cell wall carbohydrates, obtained from:
    1. CarboSource (http://www.ccrc.uga.edu/~carbosource/CSS_home.html)
    2. Plant probes (http://www.plantprobes.net/index.php)
  12. Alexa Fluor® 488 secondary antibodies (against the host of the primary antibody)
    1. Goat anti-mouse (Thermo Fisher Scientific, Invitrogen, catalog number: A-11001 )
    2. Goat anti-rat (Thermo Fisher Scientific, Invitrogen, catalog number: A-11006 )
  13. Clear nail polish
  14. Sodium phosphate, dibasic (Na2HPO4·2H2O) (Carl Roth, catalog number: 4984.2 )
  15. Sodium phosphate, monobasic (NaH2PO4·2H2O) (Carl Roth, catalog number: T879.2 )
  16. Bovine serum albumin (BSA), IgG free (Carl Roth, catalog number: 3737.2 )
  17. Pontamine S4B (sold as Direct Red 23) (Sigma-Aldrich, catalog number: 212490-50G )
  18. Sodium chloride (NaCl) (Carl Roth, catalog number: P029.1 )
  19. Phosphate-buffered saline (PBS), pH 7.0 (see Recipes)
  20. Blocking solution (see Recipes)
  21. Antibody solution (see Recipes)
  22. 0.01% (w/v) S4B in 50 mM NaCl (see Recipes)

Equipment

  1. Manual pipettes (Eppendorf, models: Research® plus and Repeater® plus ; or similar style)
  2. Water purification system (Milli-Q or similar style)
  3. Balance
  4. Autoclave
  5. Orbital shaker
  6. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 ; or similar style)
  7. Leica TCS SP8 confocal microscope (Leica Microsystems, model: TCS SP8 ) with the following key components:
    1. DM 5000 upright microscope with manual stage
    2. HC PL APO 10x/0.40 CS (Leica Microsystems, catalog number: 15506285 ) objective
    3. Compact LIAchroic AOTF unit with 488 nm and 552 nm solid state lasers
    4. SP8 Scan Head with LIAchroic beam splitters for filter-free spectral detection
      Note: Alternatively, use a conventional confocal microscope equipped with filter-based detection. Filters designed for green fluorescent proteins (GFP) and red fluorescent proteins (RFP) are generally suitable for the acquisition of Alexa Fluor® 488 and Pontamine S4B signals, respectively. In this protocol, the emission of fluorophores was detected in 500-530 nm and 590-700 nm ranges.
    5. Two fluorescence photomultipliers and one transmitted light detector
    6. Scan optics module HIVIS with rotation
  8. Computer for image acquisition and processing

Software

  1. Leica Application Suite X (LAS X) software for image acquisition
  2. Fiji is just ImageJ (Fiji) image processing package (https://fiji.sc/)

Procedure

  1. Antibody selection and ordering
    1. Based on previous publications or the WallBioNet antibody heatmap (http://glycomics.ccrc.uga.edu/wall2/antibodies/antibodyHome.html; Pattathil et al., 2010), select at least one primary antibody directed against the mucilage component of interest. Note the animal host used to produce the antibody, the polysaccharide cross-reactivity, and the epitope structure for carbohydrate antigen (if known).
      Example: CCRC-M30 is a mouse monoclonal antibody directed against Arabidopsis thaliana seed mucilage, and primarily binds this pectin-rich substrate from a diverse panel of 54 plant polysaccharides (Pattathil et al., 2010).
      Note: If using the 2F4 antibody (Liners et al., 1989) or His-tagged carbohydrate-directed modules (e.g., CBM3a; Blake et al., 2006), alternative solutions or additional labeling steps must be prepared, respectively (Voiniciuc et al., 2013 and 2015b).
    2. Order the primary antibody of interest and the corresponding Alexa Fluor® 488 secondary antibody.
      Example: For CCRC-M30, use the goat-anti-mouse antibody.
    3. Primary antibodies should be aliquoted into 1.5 ml tubes and stored at -20 °C. Once a tube is thawed, store it in the dark at 4 °C until it is completely used up.

  2. Preparing seeds for immunolabeling
    1. Label the lid of a 24-well plate with the name of the samples that will be analyzed. Always include at least one negative control.
      Example: Wild-type seeds that will be prepared alongside the samples of interest except that the primary antibody is omitted.
    2. Fill each well of the plate with 500 µl of ultrapure water (18.2 MΩ cm at 25 °C) using a repeater pipette.
    3. To each well, add around 20 Arabidopsis dry, mature seeds.
      Note: Seeds stored in small paper bags can be directly transferred to the wells, minimizing the risk of contamination. For more detailed instructions about seed harvesting and storage conditions for mucilage analysis, see the protocol by (Voiniciuc and Günl, 2016).
    4. Cover the 24-well plate with the lid and incubate for 5 min on horizontal shaker. For all shaking steps, use an orbital shaker at 100 rpm and room temperature (~23 °C).
    5. Using a 1,000 µl pipette, remove 470 µl of water from each well.
      Note: This removes the non-adherent mucilage from the seeds. Tilt the plate and pipette carefully to avoid removing any seeds along with the liquid.
    6. Add 100 µl of the fresh blocking solution using a repeater pipette.
    7. Cover the plate with the lid and shake for 30 min.
    8. Remove 100 µl of blocking solution with a 200 µl pipette.

  3. Primary antibody labeling
    1. To the ‘no primary antibody’ negative control, add 50 µl of plain antibody solution.
    2. To each sample, add 50 µl of the primary antibody diluted 1:10 in antibody solution.
      Note: Prepare a fresh master mix containing 5 µl of CCRC-M30 antibody plus 45 µl of antibody solution per sample. This dilution works well for most antibodies tested.
    3. Cover the 24-well plate with the lid and shake for 90 min.
    4. Remove the 50 µl of primary antibody solution with a 200 µl pipette.
      Note: Change tips if using two or more primary antibodies on one plate.
    5. Add 300 µl of PBS to each well using a repeater pipette.
    6. Gently mix seeds in the PBS wash solution by manually shaking the plate twice. Discard the wash solution using a plastic Pasteur pipette, without removing seeds.
    7. Perform four additional PBS washes by repeating steps C5 and C6.

  4. Secondary antibody labeling
    1. After removing the final wash solution, add 100 µl of the appropriate secondary antibody diluted 1:100 in antibody solution with a repeater pipette.
      Example: For CCRC-M30 labeling, add a freshly prepared mixture of 1 µl of Alexa Fluor® 488 goat-anti-mouse antibody and 99 µl of antibody solution to each sample.
    2. Shake the 24-well plate, covered with the lid and wrapped in aluminum foil, for 90 min.
    3. Remove the 100 µl of secondary antibody solution with a 200 µl pipette.
      Note: Change tips if using different secondary antibodies on one plate.
    4. Add 300 µl of PBS to each well using a repeater pipette.
    5. Gently mix seeds in the PBS wash solution by manually shaking the plate twice. Discard the wash solution using a plastic Pasteur pipette, without removing seeds.
    6. Perform four additional PBS washes by repeating steps D4 and D5.
    7. After the final wash, seeds can be stored in 300 µl of PBS in the dark at 4 °C overnight, or used immediately for the following steps.
      Note: Keep the seeds in the 24-well plate, until they are ready to be transferred to a glass slide for confocal imaging.

  5. Seed counterstaining (Optional)
    1. The distribution of carbohydrates epitopes in mucilage capsules is best examined relative to seed surfaces counterstained with Pontamine S4B.
      Note: The choice of the counterstain is important for the interpretation of the results. S4B specifically binds cellulose microfibrils unlike the commonly used Calcofluor stain, which also fluoresces in the presence of pectic galactan, xylan, and galactomannan (Anderson et al., 2010).
    2. To each well, add 300 µl of fresh 0.01% (w/v) Pontamine S4B in 50 mM NaCl.
    3. Shake the 24-well plate, covered with the lid and wrapped in aluminum foil, for 30 min.
    4. Remove the S4B solution with a plastic Pasteur pipette.
    5. Add 300 µl of water to each well using a repeater pipette.
    6. Gently mix seeds with the water by manually shaking the plate twice. Discard the wash solution using a plastic Pasteur pipette, without removing seeds.
    7. Perform two additional water washes by repeating steps E5 and E6.
    8. The seeds are ready to image and can be transferred to a glass slide (one per sample) using a plastic Pasteur pipette or a 1,000 µl pipette.
    9. A cover slip glass (thickness No. 1.5H) is placed over each drop containing seeds, and additional water is added (if necessary). If samples will be stored for more than 60 min, the cover slips should be sealed to glass slides with nail polish to limit evaporation.

  6. Image acquisition and processing
    1. Examine each glass slide containing whole seeds using a 10x objective equipped on a Leica TCS SP8 confocal system.
      Note: Other microscopes that can excite and detect the emission of green and red fluorescent proteins (GFP and RFP) are also suitable.
    2. Initialize the confocal hardware and the Leica Application Suite X (LAS X) software to begin the image acquisition. Turn on the 488 nm and 552 nm solid state lasers.
    3. Use transmitted light and the manual stage controls to focus on a seed of interest.
    4. Simultaneously excite Alexa Fluor® 488 and S4B using 488 nm and 552 nm solid state lasers, and acquire their signals using two fluorescence photomultipliers (PMT; PMT 1 = 500-530 nm; PMT 2 = 590-700 nm).
      Note: Only use simultaneous acquisition, if there is no significant cross-talk between two or more fluorophores. Otherwise, the confocal system should be used in sequential scanning mode. Figure 1 shows that the aforementioned parameters can separate Alexa Fluor® 488 and S4B.


      Figure 1. Excitation and emission parameters to detect two fluorophores. A-I. Single optical sections of the same region of mucilage around a wild-type (Col-0) seed immunolabelled with CCRC-M30 and counterstained with S4B. Fluorescent signals were acquired using distinct Excitation (Ex) and Emission (Em) parameters. Images were processed in Fiji. Bars = 75 µm.

    5. Briefly compare the fluorescence of the different samples and controls prepared, then set the final acquisition settings (e.g., scan format, power of each laser, gain of each detector) for the whole experiment.
      Example: Figure 2 shows the CCRC-M30 and S4B labeling of Col-0 wild-type and muci21-1 mutant seeds. In contrast to these samples, no mucilage fluorescence was detected using the same acquisition settings around wild-type seeds prepared without the CCRC-M30 primary antibody and the S4B dye.


      Figure 2. Pectin and cellulose distribution around wild-type and muci21-1 seeds. A-I. Single optical sections of three different seeds imaged using identical acquisition parameters and processed in paralleled in Fiji. The negative control (G to I) represents wild-type (Col-0) seeds incubated without the CCRC-M30 primary antibody and the S4B counterstain. Bars = 200 µm.

    6. Acquire Z-stacks of multiple seeds for each sample using identical acquisition settings. Use the scan field rotation to analyze all seeds from a similar orientation.
    7. The acquired Leica confocal images (.lif file format) can be loaded in the Fiji image processing package for processing and/or exporting images for publication (Schindelin et al., 2012).

Recipes

  1. Phosphate-buffered saline (PBS), pH 7.0
    1. Mix 5.34 g of sodium phosphate, dibasic (30 mM final concentration) and 3.12 g of sodium phosphate, monobasic (20 mM final concentration)
    2. Add 900 ml of purified water and stir to dissolve the salts
    3. Check that pH = 7.0 and adjust if necessary
    4. Fill to 1,000 ml with water, stir once again and autoclave

      Note: The sterile solution is stable for at least 1 year.

  2. Blocking solution
    1. Weigh 0.5 g of BSA in a 15 ml Falcon tube and fill to 10 ml with PBS
    2. Vortex mix until the BSA is completely dissolved

      Note: Make this solution fresh every time.

  3. Antibody solution
    1. Weigh 0.1 g of BSA in a 15 ml Falcon tube and fill to 10 ml with PBS
    2. Vortex mix until the BSA is completely dissolved

      Note: Make this solution fresh every time.

  4. 0.01% (w/v) S4B in 50 mM NaCl
    1. Dissolve 0.5 g of Pontamine S4B per 10 ml of ultrapure water to prepare a 5% (w/v) concentrated stock solution, which is stable in the dark at 4 °C for several months
    2. Dissolve 5.84 g of NaCl in 100 ml of ultrapure water, and autoclave to prepare a 100 mM NaCl concentrated stock. The sterile solution is stable for at least 1 year
    3. For each experiment, prepare a fresh 0.01% (w/v) S4B in 50 mM NaCl master mix containing 0.6 μl of 5% (w/v) of S4B, 150 μl of 100 mM NaCl and 149.4 μl of ultrapure water per sample

Acknowledgments

This protocol was briefly described by (Voiniciuc et al., 2015a), and was modified from the (Young et al., 2008; Harpaz-Saad et al., 2011) methods. I performed this work in the laboratory of Dr. Björn Usadel at Forschungzentrum Jülich with support from the Natural Sciences and Engineering Research Council of Canada (NSERC PGS-D3 grant).

References

  1. Anderson, C. T., Carroll, A., Akhmetova, L. and Somerville, C. (2010). Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots. Plant Physiol 152(2): 787-796.
  2. Blake, A. W., McCartney, L., Flint, J. E., Bolam, D. N., Boraston, A. B., Gilbert, H. J. and Knox, J. P. (2006). Understanding the biological rationale for the diversity of cellulose-directed carbohydrate-binding modules in prokaryotic enzymes. J Biol Chem 281(39): 29321-29329.
  3. Harpaz-Saad, S., McFarlane, H. E., Xu, S., Divi, U. K., Forward, B., Western, T. L. and Kieber, J. J. (2011). Cellulose synthesis via the FEI2 RLK/SOS5 pathway and cellulose synthase 5 is required for the structure of seed coat mucilage in Arabidopsis. Plant J 68(6): 941-953.
  4. Liners, F., Letesson, J. J., Didembourg, C. and Van Cutsem, P. (1989). Monoclonal antibodies against pectin: Recognition of a conformation induced by Calcium. Plant Physiol 91(4): 1419-1424.
  5. Martin, C. and Blatt, M. (2013). Manipulation and misconduct in the handling of image data. Plant Cell 25(9): 3147-3148.
  6. Pattathil, S., Avci, U., Baldwin, D., Swennes, A. G., McGill, J. A., Popper, Z., Bootten, T., Albert, A., Davis, R. H., Chennareddy, C., Dong, R., O'Shea, B., Rossi, R., Leoff, C., Freshour, G., Narra, R., O'Neil, M., York, W. S. and Hahn, M. G. (2010). A comprehensive toolkit of plant cell wall glycan-directed monoclonal antibodies. Plant Physiol 153(2): 514-525.
  7. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682.
  8. Voiniciuc, C. (2016). Quantification of the mucilage detachment from Arabidopsis seeds. Bio-protocol 6: e1802.
  9. Voiniciuc, C., Dean, G. H., Griffiths, J. S., Kirchsteiger, K., Hwang, Y. T., Gillett, A., Dow, G., Western, T. L., Estelle, M. and Haughn, G. W. (2013). Flying saucer1 is a transmembrane RING E3 ubiquitin ligase that regulates the degree of pectin methylesterification in Arabidopsis seed mucilage. Plant Cell 25(3): 944-959.
  10. Voiniciuc, C. and Günl, M. (2016) Analysis of monosaccharides in total mucilage extractable from Arabidopsis seeds. Bio-protocol 6: e1801.
  11. Voiniciuc, C., Günl, M., Schmidt, M. H. and Usadel, B. (2015a). Highly branched xylan made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 links mucilage to Arabidopsis seeds. Plant Physiol 169: 2481-2495.
  12. Voiniciuc, C., Schmidt, M. H., Berger, A., Yang, B., Ebert, B., Scheller, H. V., North, H. M., Usadel, B. and Gunl, M. (2015b). MUCILAGE-RELATED10 produces galactoglucomannan that maintains pectin and cellulose architecture in Arabidopsis seed mucilage. Plant Physiol 169(1): 403-420.
  13. Voiniciuc, C., Yang, B., Schmidt, M. H., Günl, M., Usadel, B. (2015c). Starting to gel: how Arabidopsis seed coat epidermal cells produce specialized secondary cell walls. Int J Mol Sci 16: 3452-3473.
  14. Voiniciuc, C., Zimmermann, E., Schmidt, M. H., Gunl, M., Fu, L., North, H. M. and Usadel, B. (2016). Extensive natural variation in Arabidopsis seed mucilage structure. Front Plant Sci 7: 803.
  15. Young, R. E., McFarlane, H. E., Hahn, M. G., Western, T. L., Haughn, G. W. and Samuels, A. L. (2008). Analysis of the Golgi apparatus in Arabidopsis seed coat cells during polarized secretion of pectin-rich mucilage. Plant Cell 20(6): 1623-1638.

简介

除了合成和分泌大量的果胶聚合物(Young等人,2008)外,拟南芥种皮表皮细胞产生少量的二级纤维素和半纤维素细胞壁(Voiniciuc等,,2015c)。这些组分复杂连接,并在成熟种子水合时作为大胶囊释放。在较小的粘液组分的结构中的改变可以对该凝胶状细胞壁的结构产生显着的影响。这里描述的免疫标记方案使得可以可视化种子胶囊中特定多糖的分布。

背景 自从拟南芥种皮表皮细胞(Young等人,2008)第一次富含果胶的胶质综合免疫荧光分析以来,在这种特殊的细胞壁中已经检测到另外类型的多糖(Voinicucucum,等等。,2015a; 2015b和2015c)。为了平行处理更多的样本,我修改了原始方案(在1.5 ml微量离心管中执行; Young等人,2008; Harpaz-Saad等人,2011年)到24孔板格式。我建议用Pontamine S4B(一种比以前的污渍更具体的纤维素荧光染料)来重新研磨种子(Anderson等人,2010)。通过测试多个荧光团之间的串扰,并为图像采集和处理设定明确的指导,该方案产生可重复的粘液表型,可以可靠地解释。

关键字:拟南芥, 植物细胞壁, 免疫标记, 糖类, 荧光, 种皮, 显微镜检查

材料和试剂

  1. 个人防护设备(安全眼镜,实验室外套,手套)
  2. 1.5毫升卡帽管
  3. 带盖的24孔板(VWR,目录号:734-2325)
  4. 塑料巴斯德移液器(VWR,目录号:LSUK711117S / 20)
  5. 手动移液器提示(Eppendorf,Research plus和Repeater plus或类似样式)
  6. 铝箔
  7. MARIENFELD玻片,低自发荧光,L x B x H:76 x 26 x 1 mm;预洗涤;接地边缘,90°幻灯片(VWR,目录号:631-9464)
  8. 精密盖玻片,厚度1.5H(Marienfeld-Superior,目录号:0107222)
  9. 15毫升Falcon管(VWR,目录号:734-0452)
  10. 拟南芥成熟,干种子
  11. 针对植物细胞壁碳水化合物的一抗,获自:
    1. CarboSource( http://www.ccrc.uga.edu /〜carbosource/CSS_home.html
    2. 植物探针( http://www.plantprobes.net/index.php
  12. Alexa Fluor 488次抗体(针对第一抗体的宿主)
    1. 山羊抗小鼠(Thermo Fisher Scientific,Invitrogen,目录号:A-11001)
    2. 山羊抗大鼠(Thermo Fisher Scientific,Invitrogen,目录号:A-11006)
  13. 透明指甲油
  14. 磷酸钠,二元碱(Na 2 HPO 4·2H 2 O)(Carl Roth,目录号:4984.2)
  15. 磷酸二氢钠(NaH 2 PO 4·2H 2 O)(Carl Roth,目录号:T879.2)
  16. 牛血清白蛋白(BSA),不含IgG(Carl Roth,目录号:3737.2)
  17. Pontamine S4B(以直接红23的形式出售)(Sigma-Aldrich,目录号:212490-50G)
  18. 氯化钠(NaCl)(Carl Roth,目录号:P029.1)
  19. 磷酸盐缓冲盐水(PBS),pH 7.0(参见食谱)
  20. 阻塞解决方案(见配方)
  21. 抗体溶液(参见食谱)
  22. 在50mM NaCl中的0.01%(w / v)S4B(参见食谱)

设备

  1. 手动移液器(Eppendorf,型号:Research ® plus和Repeater ® plus;或类似样式)
  2. 净水系统(Milli-Q或类似风格)
  3. 平衡
  4. 高压灭菌器
  5. 轨道摇床
  6. 涡旋搅拌机(Scientific Industries,型号:Vortex-Genie 2;或类似款式)
  7. Leica TCS SP8共焦显微镜(Leica Microsystems,型号:TCS SP8)具有以下关键部件:
    1. DM 5000立式显微镜,手动阶段
    2. HC PL APO 10x / 0.40 CS(Leica Microsystems,目录号:15506285)目标
    3. 具有488 nm和552 nm固态激光器的紧凑型LIAchroic AOTF单元
    4. SP8扫描头带有无滤光片光谱检测的LIAchroic分光器
      注意:或者,使用配有基于过滤器的检测的常规共聚焦显微镜。设计用于绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)的滤光片通常适用于获得Alexa Fluor 488和Pontamine( S4B信号。在该方案中,在500-530nm和590-700nm范围内检测到荧光团的发射。
    5. 两个荧光光电倍增管和一个透射光检测器
    6. 扫描光学模块艾滋病病毒与旋转
  8. 用于图像采集和处理的计算机

软件

  1. 用于图像采集的Leica Application Suite X(LAS X)软件
  2. 斐济只是ImageJ(斐济)图像处理包( https://fiji.sc/

程序

  1. 抗体选择和排序
    1. 基于以前的出版物或WallBioNet抗体热图( http: //glycomics.ccrc.uga.edu/wall2/antibodies/antibodyHome.html ; Pattathil等人,2010),选择至少一种针对感兴趣的粘液成分的一级抗体。注意用于产生抗体的动物宿主,多糖交叉反应性和碳水化合物抗原的表位结构(如果已知)。
      实施例:CCRC-M30是针对拟南芥种子粘液的小鼠单克隆抗体,并且主要从54种植物多糖的不同组合中结合富含果胶的底物(Pattathil等人,2010)。
      注意:如果使用2F4抗体(Liners等人,1989)或His标记的碳水化合物定向模块(例如,CBM3a; Blake等人,2006),则必须分别制备替代溶液或附加标记步骤(Voiniciuc et al。,2013和2015b)。
    2. 订购感兴趣的第一抗体和相应的Alexa Fluor 488二抗。
      例如:对于CCRC-M30,使用山羊抗小鼠抗体。
    3. 初级抗体应分装在1.5 ml管中,储存于-20°C。一旦管被解冻,将其在黑暗中保存在4°C,直到其完全用完。

  2. 准备种子进行免疫标记
    1. 将24孔板的盖子标记为将要分析的样品的名称。始终至少包含一个阴性对照。
      实施例:除了一级抗体被省略之外,将与感兴趣的样品一起制备的野生型种子。
    2. 使用中继器移液管,用500μl超纯水(25°C时为18.2MΩcm)将板的每个孔填充。
    3. 对每个孔,加入约20个拟南芥干/干成熟种子。
      注意:存储在小纸袋中的种子可以直接转移到井中,从而最大限度地减少污染的风险。有关种子收获和粘液分析储存条件的更详细说明,请参阅(Voiniciuc和Günl,2016)的方案。
    4. 用盖盖住24孔板,并在卧式摇床上孵育5分钟。对于所有的振动步骤,使用轨道振荡器在100 rpm和室温(〜23°C)。
    5. 使用1000μl移液管,从每个孔中清除470μl的水。
      注意:这从种子中除去非粘附的粘液。仔细倾斜板和移液器,以避免与液体一起除去任何种子。
    6. 使用中继器移液管加入100μl新鲜的阻塞溶液。
    7. 盖上盖子摇匀30分钟
    8. 用200μl移液管清除100μl封闭溶液。

  3. 一抗标记
    1. 对“无一抗”阴性对照,加入50μl纯抗体溶液。
    2. 向每个样品中加入50μl抗体溶液中1:10稀释的一抗。
      注意:准备一份含有5μlCCRC-M30抗体的新鲜主混合物,每个样品加上45μl抗体溶液。对于大多数测试的抗体,这种稀释效果很好。
    3. 用盖盖住24孔板,摇动90分钟。
    4. 用200μl移液管取出50μl的一级抗体溶液。
      注意:如果在一个平板上使用两个或更多个主要抗体,请更改提示。
    5. 使用中继器移液管向每个孔中加入300μlPBS。
    6. 通过手动摇动板两次,轻轻地将种子混合在PBS洗液中。使用塑料巴斯德移液器丢弃洗涤液,不要去除种子。
    7. 通过重复步骤C5和C6进行四次PBS洗涤。

  4. 二次抗体标记
    1. 除去最终的洗涤溶液后,加入100μl用抗体溶液1:100稀释的适当的二抗。
      实施例:对于CCRC-M30标记,加入新鲜制备的1μlAlexa Fluor的混合物488山羊抗小鼠 抗体和99μl抗体溶液。
    2. 摇动24孔板,盖上盖子并裹在铝箔上90分钟
    3. 用200μl移液管取出100μl二抗溶液。
      注意:如果在一块板上使用不同的二次抗体,请更改提示。
    4. 使用中继器移液管向每个孔中加入300μlPBS。
    5. 通过手动摇动板两次,轻轻地将种子混合在PBS洗液中。使用塑料巴斯德移液器丢弃洗涤液,不要去除种子。
    6. 通过重复步骤D4和D5进行四次PBS洗涤。
    7. 最后一次洗涤后,种子可以在4℃的黑暗中保存在300μl的PBS中过夜,或者立即用于下列步骤。
      注意:将种子保留在24孔板中,直到准备好转移到玻璃片上进行共焦成像。

  5. 种子复染(可选)
    1. 相对于用Pontamine S4B复染的种子表面,最好检查胶体胶囊中碳水化合物表位的分布。
      注意:对于结果的解释,重新选择复数是很重要的。 S4B特异性结合纤维素微原纤维不同于通常使用的Calcofluor染色剂,其在果胶半乳聚糖,木聚糖和半乳甘露聚糖存在下也发荧光(Anderson等人,2010)。
    2. 向每个孔中加入300μl新鲜的0.01%(w / v)Pontamine S4B在50mM NaCl中
    3. 摇动24孔板,盖上盖子并裹上铝箔30分钟
    4. 用塑料巴斯德吸管移除S4B溶液。
    5. 使用中继器移液管向每个孔中加入300μl水。
    6. 用水轻轻搅拌种子两次。使用塑料巴斯德移液器丢弃洗涤液,不要去除种子。
    7. 通过重复步骤E5和E6进行两次额外的水洗。
    8. 种子准备成像,可以使用塑料巴斯德吸管或1,000μl移液管将其转移到载玻片(每个样品一个)。
    9. 将盖玻片(厚度1.5H)放置在每个含有种子的液滴上,并加入额外的水(如果需要)。如果样品储存时间超过60分钟,盖玻片应用指甲油密封到玻璃片上,以限制蒸发。

  6. 图像采集和处理
    1. 使用配备在Leica TCS SP8共焦系统上的10x物镜检查含有全种子的每个载玻片。
      注意:可以激发和检测绿色和红色荧光蛋白(GFP和RFP)发射的其他显微镜也是合适的。
    2. 初始化共焦硬件和Leica Application Suite X(LAS X)软件,开始图像采集。打开488 nm和552 nm固体激光器。
    3. 使用透射光和手动舞台控制来关注感兴趣的种子。
    4. 使用488nm和552nm固态激光器同时激发Alexa Fluor <48>和48B,并使用两个荧光光电倍增管(PMT; PMT 1 = 500-530nm; PMT 2 = 590-700纳米)。
      注意:如果两个或更多个荧光团之间没有明显的串扰,则仅使用同时采集。否则,共焦系统应该在顺序扫描模式下使用。图1显示,上述参数可以分开Alexa Fluor 488和S4B。


      图1.检测两个荧光团的激发和发射参数。 A-I。在与CCRC-M30免疫标记的野生型(Col-0)种子周围的相同粘液区域的单一光学切片并用S4B复染色。使用不同的激发(Ex)和发射(Em)参数获得荧光信号。图像在斐济处理。棒=75μm。

    5. 简单比较准备的不同样品和对照的荧光,然后设置整个实验的最终采集设置(例如,扫描格式,每个激光的功率,每个检测器的增益)。
      实施例:图2显示了Col-0野生型和muci21-1突变种子的CCRC-M30和S4B标记。与这些样品相反,使用在不含CCRC-M30一抗和S4B染料的情况下制备的野生型种子周围的相同采集设置,也没有检测到粘液荧光。


      图2.野生型和muci21-1种子周围的果胶和纤维素分布。A-I。使用相同采集参数成像并在斐济平行处理的三种不同种子的单个光学部分。阴性对照(G至I)表示不含CCRC-M30一抗和S4B复染色培养的野生型(Col-0)种子。酒吧= 200μm。

    6. 使用相同的采集设置为每个样品获取多个种子的Z-堆叠。使用扫描场旋转来分析来自类似方向的所有种子。
    7. 获得的Leica共焦图像(.lif文件格式)可以加载在斐济图像处理包中,用于处理和/或导出出版图像(Schindelin等人,2012年)。

食谱

  1. 磷酸缓冲盐水(PBS),pH 7.0
    1. 混合5.34g磷酸钠,二元(30mM终浓度)和3.12g磷酸钠,一元(20mM终浓度)
    2. 加入900ml纯净水,搅拌溶解盐
    3. 检查pH = 7.0,如有必要,进行调整
    4. 用水填充至1000毫升,再次搅拌,然后高压灭菌

      注意:无菌解决方案至少保持1年。

  2. 阻塞解决方案
    1. 称量0.5g BSA在15ml Falcon管中,并用PBS将其填充至10ml
    2. 涡旋混合,直到BSA完全溶解

      注意:每次都会使此解决方案变得新鲜。

  3. 抗体溶液
    1. 称取0.1g BSA在15ml Falcon管中,并用PBS将其填充至10ml
    2. 涡旋混合,直到BSA完全溶解

      注意:每次都会使此解决方案变得新鲜。

  4. 在50mM NaCl中的0.01%(w / v)S4B
    1. 每10ml超纯水溶解0.5 g的Pontamine S4B,以制备5%(w / v)浓缩的储备溶液,该溶液在黑暗中在4℃下稳定数月。
    2. 将5.84g NaCl溶于100ml超纯水中,高压釜中制备100mM NaCl浓缩液。无菌溶液至少稳定1年
    3. 对于每个实验,在含有0.6μl5%(w / v)的S4B,150μl100mM NaCl和149.4μl超纯水的50mM NaCl主混合物中制备新鲜的0.01%(w / v) br />

致谢

该方案由(Voiniciuc等人,2015a)简要描述,并且由(Young等人,2008; Harpaz-Saad等人,2011)进行了修改) 方法。在加拿大自然科学与工程研究理事会(NSERC PGS-D3授权)的支持下,我在ForschungzentrumJülich博士BjörnUsadel博士的实验室进行了这项工作。

参考

  1. 安德森,CT,Carroll,A.,Akhmetova,L.和Somerville,C。(2010)。在拟南芥根中细胞壁扩增期间纤维素重新取向的实时成像。植物生理152(2): 787-796。
  2. Blake,AW,McCartney,L.,Flint,JE,Bolam,DN,Boraston,AB,Gilbert,HJ和Knox,JP(2006)。了解原核生物酶中纤维素指导的碳水化合物结合模块多样性的生物学原理。 / em> 281(39):29321-29329。
  3. Harpaz-Saad,S.,McFarlane,HE,Xu,S.,Divi,UK,Forward,B.,Western,TL and Kieber,JJ(2011)。通过FEI2 RLK / SOS5途径的纤维素合成和纤维素合成酶5是拟南芥中种皮粘液结构所必需的, / em>。植物J 68(6):941-953。
  4. Liners,F.,Letesson,JJ,Didembourg,C.and Van Cutsem,P。(1989)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov / pubmed / 16667195“target =”_ blank“>针对果胶的单克隆抗体:由钙诱导的构象的识别植物生理91(4):1419-1424。
  5. Martin,C. and Blatt,M。(2013)。&nbsp; 处理图像数据时的操纵和不当行为植物细胞 25(9):3147-3148。
  6. Pattathil,S.,Avci,U.,Baldwin,D.,Swennes,AG,McGill,JA,Popper,Z.,Bootten,T.,Albert,A.,Davis,RH,Chennareddy,C.,Dong,R ,O'Shea,B.,Rossi,R.,Leoff,C.,Freshour,G.,Narra,R.,O'Neil,M.,York,WS and Hahn,MG(2010)。&nbsp; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/20363856”target =“_ blank”>植物细胞壁聚糖指导的单克隆抗体的综合工具包。 a>植物生理 153(2):514-525。
  7. Schindelin,J.,Arganda-Carreras,I.,Frize,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B 。,Tinevez,JY,White,DJ,Hartenstein,V.,Eliceiri,K.,Tomancak,P。和Cardona,A.(2012)。&nbsp; 斐济:用于生物图像分析的开源平台。 Nat方法 9(7 ):676-682。
  8. Voiniciuc,C。(2016)。定量从拟南芥种子生物协议 6:e1802。
  9. Voiniciuc,C.,Dean,GH,Griffiths,JS,Kirchsteiger,K.,Hwang,YT,Gillett,A.,Dow,G.,Western,TL,Estelle,M.and Haughn,GW(2013) 飞碟1是跨膜RING E3泛素连接酶,其调节程度拟南芥种子胶浆中的果胶甲基酯化作用。植物细胞 25(3):944-959。
  10. Voiniciuc,C.和Günl,M。(2016)&nbsp; 单糖分析从拟南芥种子中提取的总粘液生物协议 6:e1801。
  11. Voiniciuc,C.,Günl,M.,Schmidt,MH and Usadel,B。(2015a)。&lt; a class =“ke-insertfile”href =“https://www.ncbi.nlm.nih.gov/ pubmed / 26482889“target =”_ blank“>由IRREGULAR XYLEM14和MUCILAGE-RELATED21制备的高度分枝的木聚糖将粘液连接到拟南芥种子。植物生理学169:2481 -2495。
  12. Voiniciuc,C.,Schmidt,MH,Berger,A.,Yang,B.,Ebert,B.,Scheller,HV,North,HM,Usadel,B.and Gunl,M。(2015b)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26220953”target =“_ blank”> MUCILAGE-RELATED10产生在拟南芥中维持果胶和纤维素结构的半乳糖甘露聚糖/ em>种子粘液。植物生理学169(1):403-420。
  13. Voiniciuc,C.,Yang,B.,Schmidt,MH,Günl,M.,Usadel,B。(2015c)。&lt; a class =“ke-insertfile”href =“https://www.ncbi.nlm .nih.gov / pubmed / 25658798“target =”_ blank“>开始凝胶:拟南芥种皮表皮细胞如何产生专门的二次细胞壁 Int J Mol Sci < / em> 16:3452-3473。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Voiniciuc, C. (2017). Whole-seed Immunolabeling of Arabidopsis Mucilage Polysaccharides. Bio-protocol 7(11): e2323. DOI: 10.21769/BioProtoc.2323.
  2. Voiniciuc, C., Gunl, M., Schmidt, M. H. and Usadel, B. (2015). Highly Branched Xylan Made by IRREGULAR XYLEM14 and MUCILAGE-RELATED21 Links Mucilage to Arabidopsis Seeds. Plant Physiol 169(4): 2481-2495.
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