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Oct 2019
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Histochemical Staining of Suberin in Plant Roots
植物根部软木脂的组织化学染色   

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Abstract

Histological stains are useful tools for characterizing cell shape, arrangement and the material they are made from. Stains can be used individually or simultaneously to mark different cell structures or polymers within the same cells, and to visualize them in different colors. Histological stains can be combined with genetically-encoded fluorescent proteins, which are useful for understanding of plant development. To visualize suberin lamellae by fluorescent microscopy, we improved a histological staining procedure with the dyes Fluorol Yellow 088 and aniline blue. In the complex plant organs such as roots, suberin lamellae are deposited deep within the root on the endodermal cell wall. Our procedure yields reliable and detailed images that can be used to determine the suberin pattern in root cells. The main advantage of this protocol is its efficiency, the detailed visualization of suberin localization it generates in the root, and the possibility of returning to the confocal images to analyze and re-evaluate data if necessary.

Keywords: Suberin (软木脂), Fluorol Yellow (荧光黄), Root (根), Confocal microscopy (共聚焦显微镜)

Background

Suberin is a complex polyester that, together with cutin and lignin, forms a physical barrier in land plants. In Arabidopsis (Arabidopsis thaliana), suberin primarily accumulates in the cell walls of epidermal leaf cells, in the seed coat, in the periderm and in the cell wall of root endodermal cells. In the root, suberin deposition is dynamic process (Barberon et al., 2016; Andersen et al., 2018; Ursache et al., 2020) that is modulated by various nutritional stresses, drought stress, and phytohormone signaling pathways (Baxter et al., 2009; Kosma et al., 2014; Barberon et al., 2016). Suberin in root endodermal cells acts as a barrier that controls the uptake of water and nutrients (Baxter et al., 2009; Krishnamurthy et al., 2009 and 2011; Wang et al., 2019), but also provides a protective layer against various pathogens (Thomas et al., 2007; Ranathunge et al., 2008; Holbein et al., 2019; Zhou et al., 2020; Emonet et al., 2020). Moreover, suberin is also involved in periderm formation, the tissue that envelops secondary stems as part of the bark. Given its position deep within the root, it is technically challenging to study suberin. Developing a reliable tool to study the multifunctional activities of suberin and its precise localization in cells would contribute to new discoveries in many areas of plant research. A protocol for suberin staining was described previously by Lux et al. (2005) and subsequently modified over the years (Naseer et al., 2012; Fujita et al., 2020; Ursache et al., 2020). Here, we provide a standardized protocol to stain suberin and to acquire data from 10-15 roots at a single tile-scan image by confocal microscopy in a short time. This protocol will enhance the efficiency of data generation with possibility to re-analyze data if necessary.


Materials and Reagents

  1. Square dishes 120 × 120 × 17 mm (Greiner Bio-One GmbH, catalog number: 688102)

  2. Flat-bottom 6-, 12-, and 24-well plates with lid (Stemcell Technology, catalog number: 100-0096)

  3. Adhesive or any available tape

  4. 2 ml Eppendorf tubes

  5. Falcon tubes 15 or 50 ml (Stemcell Technology, catalog numbers: 38010, 100-0090; 38009, 100-0092)

  6. Aluminum foil

  7. Parafilm “M” (Merck, catalog number: P7543-1EA)

  8. SuperFrost microscope slides (Thermo Scientific, catalog number: 12362098)

  9. Coverslips (Thermo Scientific, catalog number: 12393138)

    Note: Material needed for the protocol is illustrated in Figure 1.

  10. Chambered cover glass (VWR, chamber slides-one chamber, NuncTM Lab-TekTM, catalog number: 734-2056)

  11. Arabidopsis seeds

    GROWTH CONDITIONs: Sow surface-sterilized Arabidopsis seeds on half-strength MS agar square plates (45.5 ml). Stratify plates for 2 days at 4 °C in the dark and then transfer to growth chambers under a 16-h-light/8-h-dark photoperiod at 21 °C. Allow seedlings to grow on vertically-oriented plates for 5 days.

    Note: 5-day old seedlings are the standard age of seedlings in our experimental setups. The age of seedlings can be adapted according to experiments.

  12. Milli-Q Water (H2O)

  13. Sucrose (VWR International, catalog number: 27483.294)

  14. Murashige and Skoog basal salt mixture (MS salts) (Duchefa Biochemie, catalog number: M0221.0050)

  15. 2-[N-morpholino] ethanesulfonic acid (MES) (Duchefa Biochemie, catalog number: M1503.0100)

  16. Agar (Lab M, catalog number: MC029)

  17. Potassium hydroxide (KOH) (Merck KGaA, catalog number: 1.05021.1000)

  18. Ethanol (EtOH) (Sigma-Aldrich, catalog number: 32221-2.5L)

  19. Lactic acid (Honeywell, FlukaTM, catalog number: 15636730)

  20. Fluorol Yellow 088 (Santa Cruz Biotechnology, catalog number: 81-37-8)

  21. Aniline blue (Sigma-Aldrich, catalog number: 66687-07-8), 0.5% in MilliQ Water

  22. Seed sterilization with ethanol (see Recipes)

  23. Half-strength MS medium (see Recipes)

  24. 0.03% Fluorol Yellow 088 solution (see Recipes)

  25. 0.5% Aniline blue solution (see Recipes)



    Figure 1. Required material and equipment. A. Five-day-old Arabidopsis seedlings. Scale bar: 1 cm. B. Materials needed to stain samples. C. Dissolving Fluorol Yellow 088 in lactic acid in a 50 ml Falcon tube attached to a vortex mixer with adhesive tape. D. Dissolved Fluorol Yellow (left) and aniline blue (right) solutions.

Equipment

  1. Growth chamber to grow plant materia (Percival, CLF, Plant Climatics, AR75-L3)

  2. Vortex mixer (Instrumentfirman Labora, model: Vortex-Genie 2)

  3. Water bath (Fisher Scientific)

  4. Soft stainless steel tweezers (3B DISSECTION SOFT TWEEZERS, LABWORLD)

  5. Inverted confocal microscope (Zeiss, model: LSM880)

    Note: A fully motorized X, Y, Z scanning stage is required to perform tile scan image acquisition experiments.

  6. Objectives: 10× or 20× (suitable for monitoring the whole specimen or root, e.g., Figure 3C). For higher resolution images, a higher magnification objective, such as 40× or 63×, is required (Figure 3D)

  7. Fluorescence signal detection system for GFP and other fluorescent reporters (Shaner et al., 2007)

Software

  1. Software operating the confocal microscope

  2. ImageJ (Abràmoff et al., 2004)

  3. Microsoft Excel

  4. Prism (GraphPad)

Procedure

  1. Sample staining

    1. Add 15-20 seedlings into each well containing 2 ml of Fluorol Yellow 088 (FY088) (e.g., 12-well plate, depending on the number of samples) (Figure 2A).

    2. Close the lid and seal the plate with parafilm. Place the plate in a water bath on an aluminum foil raft at 70 °C for 20 min (Figures 2B-2D).

      Note: Bend the edges of the aluminum foil to prevent sinking. During the incubation time in the FY088 solution, prepare the 0.5% aniline blue solution and keep in the dark at room temperature until use.

    3. Transfer the seedlings from the FY088 solution to a new 12-well plate containing 2 ml water and incubate for 1 min at room temperature (Figure 2E).

    4. Remove water by pipetting and add 2 ml of 0.5% aniline blue solution to each sample (Figure 2F). Incubate for 20 min in the dark at room temperature.

    5. After incubation in aniline blue, move the seedlings to a new -, 12-, and 24-well plates containing 2-3 ml water (Figure 2G) and incubate for 10 min in the dark.



      Figure 2. Sample Staining. A. Twelve-well plate containing Arabidopsis seedlings in 2 ml FY088 solution. B. Plate sealed with parafilm. C. Sealed well plate placed on an aluminum foil raft. D. Samples placed into a water bath at 70 °C for 20 min. E. Seedlings washed in 2 ml of water. F. Well plate containing 2 ml aniline blue solution with seedlings. G. Wash aniline blue in 2 ml water. H. Preparation of microscope slide with parafilm strips to make a “spacer”.


  2. Sample preparation/mounting

    1. Cut two strips of parafilm and wrap 2-3 times around both ends of the microscope slide (Figures 2H and 3A).

    2. Add a few drops of water (30-50 μl) in the center of the slide between two parafilm strips. Position the seedlings onto this liquid using tweezers and cover with a cover glass slide (Figures 3A and 3B).

    3. For instructions on using a one-well chambered cover glass and positioning the seedlings, please follow the protocol described previously: Marhavý and Benková, (2015).


  3. Sample imaging

    1. Prepare an inverted confocal microscope for use; set lasers (for GFP: 488 nm excitation, emission 500-550 nm) and objectives.

      Note: For a broad overview of suberin localization in the root, we recommend performing tile scan imaging using either a 10× or 20× objective (in dry or water-immersion mode). For higher resolution images, we recommend using a 40× or 63× objective immersed in water or oil.

      Parameters for image capture on a Zeiss LSM 880 shown in Figure 3C: Image size – x: 21793.72 µm, y: 18702.12 µm; Scaling X – 1510 µm, Scaling Y – 1510 µm; Tile scan – tiles1, overlap in percent: 20.0, tiling mode: rectangular grid (stitched image); scan mode – plane, zoom 1.1; Pixel dwell – 0.85 µs; master gain – 482; digital gain – 1.5; digital offset – 0.00; filters – SP 555; beam splitters – MBS: MBS 488 nm, MBS_InVis: plate, FW1: Rear.

      Note: Parameters should be adjusted according to the specimen used.

    2. Mount a glass slide or chamber with seedlings and place on the stage of the confocal microscope.

    3. Activate the position list and tile scan (25 × 34 tiles) with 20% overlap between images.

      Notes:

      1. Adjust the number of tiles according to the approximate scanning area. Alternatively, activate automatic stitching (this can also be done later using the confocal software).

      2. Parameters should be adjusted according to the size of the samples.

    4. Find the root closest to the central point of the scanning area and mark the position.

    5. Deactivate the position and start scanning.

      Notes:

      1. With the above-mentioned parameters, it will take approximately 15 min to obtain the final image. This provides enough time to prepare the next sample, as described above.

      2. If the laboratory does not have suitable confocal microscope, images can be taken by a fully motorized Fluorescence stereo microscope with a tile scan option.

    6. Export confocal images in TIFF or JPEG format. Open images in ImageJ or other software and analyze your images (Figures 3C, 3D).

      Note: Be exact in terms of both the timing and procedure for each step to ensure reproducibility of your results.



      Figure 3. Sample preparation and imaging. A. A 30 μl drop of water placed on the microscope slide between two parafilm strips. B. Place 10-15 seedlings onto each slide and cover with a cover slip. C. Tile scan of stained Arabidopsis seedlings. Yellow fluorescence indicates suberin deposition in the root. The white frame surrounds the area that is expanded in D for a detailed image of suberin lamellae. Scale bar: 1 mm. D. Detailed image of suberin lamellae in endodermal root cells. Scale bar: 100 μm.

Data analysis

Open image in ImageJ Set scale; Known distance; Unit of length (mm or cm); click/mark Global and press OK. Go to ImageJ, select drawing line (segmented or freehand line) and draw a line along the root, from base to tip, press “M” to measure root length. Repeat the procedure to measure the length of the suberized region of the root. Copy the results to Excel or Prisma GraphPad for further processing (for example, to calculate suberin occupancy in the root).

Note: We do not recommend analyzing FY fluorescence intensity, since this can be influenced by discrepancies in staining time, washing, and/or imaging.

Recipes

  1. Seed sterilization with ethanol

    Transfer seeds to 2 ml Eppendorf tubes (maximum volume of seeds to be sterilized per tube should not exceed 3 mm from the bottom of the tube)

    Add 1 ml 70% EtOH, shake for 5 s and allow seeds to settle for 10 min

    Dry seeds under the clean bench

  2. Half-strength MS medium (1 L)

    Combine 10 g sucrose, 2.3 g MS salts, and 0.5 g MES, adjust the pH to 5.9 with KOH, add 8 g agar and fill with ddH2O to 1 L

    Autoclave medium for 20-30 min, and allow to cool to 55 °C before pouring plates

  3. 0.03% Fluorol Yellow 088 solution

    Fluorol Yellow 088 in lactic acid at room temperature (Figures 1C, 1D) must be freshly prepared before each experiment

    Note: To speed up dissolving FY 088 in lactic acid, attach a 50 ml Falcon tube to a vortex mixer with adhesive tape and vortex for 10-15 min (Figure 1C).

  4. 0.5% Aniline blue solution

    Aniline blue in Milli-Q Water

    Store in a dark place at room temperature

Acknowledgments

We are thankful to Niko Geldner for all the discussion and feedback to develop this protocol. This work was supported by the Swedish research Council a Starting grant to Peter Marhavý. Shahid Siddique was supported by a grant from the United States Department of Agriculture (project no. CA-D-ENM-2562-RR).

The protocol was improved based on previously published work of Lux et al. (2005).

Competing interests

There are no conflicts of interest or competing interest.

References

  1. Abràmoff, M. D., Magalhães, P. J. and Ram, S. J. (2004). Image processing with ImageJ. Biophotonics Int 11(7): 36-43.
  2. Andersen, T. G., Naseer, S., Ursache, R., Wybouw, B., Smet, W., De Rybel, B., Vermeer, J. E. M. and Geldner, N. (2018). Author Correction: Diffusible repression of cytokinin signalling produces endodermal symmetry and passage cells. Nature 559(7714): E9.
  3. Barberon, M., Vermeer, J. E., De Bellis, D., Wang, P., Naseer, S., Andersen, T. G., Humbel, B. M., Nawrath, C., Takano, J., Salt, D. E. and Geldner, N. (2016). Adaptation of Root Function by Nutrient-Induced Plasticity of Endodermal Differentiation. Cell 164(3): 447-459.
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  6. Fujita, S., De Bellis, D., Edel, K. H., Koster, P., Andersen, T. G., Schmid-Siegert, E., Denervaud Tendon, V., Pfister, A., Marhavy, P., Ursache, R., Doblas, V. G., Barberon, M., Daraspe, J., Creff, A., Ingram, G., Kudla, J. and Geldner, N. (2020). SCHENGEN receptor module drives localized ROS production and lignification in plant roots. EMBO J 39(9): e103894.
  7. Holbein, J., Franke, R. B., Marhavy, P., Fujita, S., Gorecka, M., Sobczak, M., Geldner, N., Schreiber, L., Grundler, F. M. W. and Siddique, S. (2019). Root endodermal barrier system contributes to defence against plant-parasitic cyst and root-knot nematodes. Plant J 100(2): 221-236.
  8. Kosma, D. K., Murmu, J., Razeq, F. M., Santos, P., Bourgault, R., Molina, I. and Rowland, O. (2014). AtMYB41 activates ectopic suberin synthesis and assembly in multiple plant species and cell types. Plant J 80(2): 216-229.
  9. Krishnamurthy, P., Ranathunge, K., Franke, R., Prakash, H. S., Schreiber, L. and Mathew, M. K. (2009). The role of root apoplastic transport barriers in salt tolerance of rice(Oryza sativa L.). Planta 230(1): 119-134.
  10. Krishnamurthy, P., Ranathunge, K., Nayak, S., Schreiber, L. and Mathew, M. K. (2011). Root apoplastic barriers block Na+ transport to shoots in rice(Oryza sativa L.). J Exp Bot 62(12): 4215-4228.
  11. Lux, A., Morita, S., Abe, J. and Ito, K. (2005). An improved method for clearing and staining free-hand sections and whole-mount samples. Ann Bot 96(6): 989-996.
  12. Marhavý, P. and Benková, E. (2015). Real-time Analysis of Lateral Root Organogenesis in Arabidopsis. Bio-protocol 5(8): e1446.
  13. Naseer, S., Lee, Y., Lapierre, C., Franke, R., Nawrath, C. and Geldner, N. (2012). Casparian strip diffusion barrier in Arabidopsis is made of a lignin polymer without suberin. Proc Natl Acad Sci U S A 109: 10101-10106.
  14. Ranathunge, K., Thomas, R. H., Fang, X., Peterson, C. A., Gijzen, M. and Bernards, M. A. (2008). Soybean root suberin and partial resistance to root rot caused by Phytophthora sojae. Phytopathology 98(11): 1179-1189.
  15. Shaner, N. C., Patterson, G. H. and Davidson, M. W. (2007). Advances in fluorescent protein technology. J Cell Sci (24): 4247-4260.
  16. Thomas, R., Fang, X., Ranathunge, K., Anderson, T. R., Peterson, C. A. and Bernards, M. A. (2007). Soybean root suberin: anatomical distribution, chemical composition, and relationship to partial resistance to Phytophthora sojae. Plant Physiol 144(1): 299-311.
  17. Ursache, R., De Jesus Vieira-Teixeira, C., Tendon, V. D., Gully, K., De Bellis, D., Schmid-Siegert, E., Andersen, T, G., Shekhar, V., Calderon, S., Pradervand, S., Nawrath, C., Geldner, N. and Vermeer, J. E. M. (2020). GDSL-domain containing proteins mediate suberin biosynthesis and degradation, enabling developmental plasticity of the endodermis during lateral root emergence. bioRxiv. doi: https://doi.org/10.1101/2020.06.25.171389.
  18. Wang, P., Calvo-Polanco, M., Reyt, G., Barberon, M., Champeyroux, C., Santoni, V., Maurel, C., Franke, R. B., Ljung, K., Novak, O., Geldner, N., Boursiac, Y. and Salt, D. E. (2019). Surveillance of cell wall diffusion barrier integrity modulates water and solute transport in plants. Sci Rep 9(1): 4227.
  19. Zhou, F., Emonet, A., Denervaud Tendon, V., Marhavy, P., Wu, D., Lahaye, T. and Geldner, N. (2020). Co-incidence of Damage and Microbial Patterns Controls Localized Immune Responses in Roots. Cell 180(3): 440-453 e418.

简介

[摘要]组织学染色是表征细胞形状,排列和其制成材料的有用工具。可以单独或同时使用污渍来标记同一细胞内的不同细胞结构或聚合物,并以不同的颜色对其进行可视化。组织学染色剂可以与遗传编码的荧光蛋白结合使用,这对于理解植物的发育非常有用。为了通过荧光显微镜观察木栓质片状细胞,我们改进了荧光黄088和苯胺蓝染料的组织学染色程序。在复杂的植物器官中,例如根部,木栓质薄片沉积在根部内的内胚层细胞壁上。我们的程序产生了可靠而详细的图像,可用于确定根细胞中的suberin模式。此协议的主要优点是它的效率,木栓质本地化的详细可视化它产生的根源,并返回到共焦图象进行分析和重新的可能性-评估数据如果必要的话。

[背景] Suberin是一种复杂的聚酯,与角质和木质素一起在陆地植物中形成物理屏障。在拟南芥(Arabidopsis thaliana )中,木栓质主要积聚在表皮叶细胞的细胞壁,种皮,周皮和根部内胚层细胞的细胞壁中。从根本上说,木栓质沉积是动态过程(Barberon等,2016; Andersen等,2018; Ursache等,2020),其受各种营养胁迫,干旱胁迫和植物激素信号传导途径的调节(Baxter等)。等人,2009; Kosma等人,2014; Barberon等人,2016)。根部内胚层细胞中的Suberin是控制水分和养分吸收的屏障(Baxter等,2009; Krishnamurthy等,2009和2011; Wang等,2019),但也提供了针对各种水分的保护层病原体(Thomas等,2007; Ranathunge等,2008; Holbein等,2019; Zhou等,2020; Emonet等,2020)。此外,木栓质还参与了皮层的形成,该组织包裹着次生茎,成为树皮的一部分。鉴于其在根部内的深处,研究木栓质在技术上具有挑战性。开发可靠的工具来研究木脂蛋白的多功能活性及其在细胞中的精确定位将有助于植物研究许多领域的新发现。Luxer等人先前曾描述过木栓蛋白染色的方案。(2005 ),并且随后多年来改性(纳瑟尔等人,2012; Fujit一个。等人,2020; Ursache 。等人,2020)。在这里,我们提供了一种标准化协议,可以在短时间内通过共聚焦显微镜对苏木精染色并从单个瓷砖扫描图像的10-15个根中获取数据。该协议将提高数据生成的效率,并在必要时可以重新分析数据。

关键字:软木脂, 荧光黄, 根, 共聚焦显微镜

材料和试剂
1.方盘120 × 120 × 17 mm(Greiner Bio-One GmbH,目录号:688102)     
2.带盖的平底6孔,12孔和24孔板(Stemcell Technology,目录号:100-0096)     
3.胶粘剂或任何可用的胶带     
4. 2毫升Eppendorf管     
5. 15或50 ml的猎鹰管(Stemcell Technology,目录号:38010,100-0090; 38009,100-0092)     
6.铝箔     
7.封口膜“ M”(默克,目录号:P7543-1EA)     
8. SuperFrost显微镜载玻片(Thermo Scientific,目录号:12362098 )     
9. Coverslips(Thermo Scientific,目录号:12393138)     
注意:协议所需的材料如图1所示。

10.夹室玻璃盖(VWR,腔室一体式腔室,Nunc TM Lab-Tek TM ,目录号:734-2056) 
11.拟南芥种子 
生长条件:在半强度MS琼脂方形平板(45.5 ml)上播种表面灭菌的拟南芥种子。在黑暗中于4°C下将板分层2天,然后在21°C在16小时光照/ 8小时黑暗的光周期下转移至生长室。让幼苗在垂直放置的板上生长5天。

注意:5天大的幼苗是我们实验设置中幼苗的标准年龄。幼苗的年龄可以根据实验进行调整。

12. Milli-Q水(H 2 O) 
13.蔗糖(VWR International,目录号:27483.294) 
14. Murashige和Skoog基础盐混合物(MS盐)(Duchefa Biochemie,目录号:M0221.0050) 
15. 2- [N-吗啉代]乙磺酸(MES)(Duchefa Biochemie,目录号:M1503.0100) 
16.琼脂(实验室M,目录号:MC029) 
17.氢氧化钾(Merck KGaA,目录号:1.05021.1000)   
18.乙醇(EtOH)(Sigma-Aldrich,目录号:32221-2.5L) 
19.乳酸(Honeywell,Fluka TM ,目录号:15663630) 
20.氟黄088(圣克鲁斯生物技术,目录号:81-37-8) 
21.苯胺蓝(Sigma-Aldrich,目录号:66687-07-8),在MilliQ Water中为0.5% 
22.用乙醇对种子进行灭菌(参见食谱) 
23.半强度MS培养基(请参见食谱) 
24. 0.03%氟黄088溶液(请参阅食谱) 
25. 0.5%苯胺蓝溶液(请参见食谱) 



图1.所需的材料和设备。A.五天大的拟南芥幼苗。比例尺:1厘米。B.染色样品所需的材料。C.在附有涡流混合器的50ml Falcon管中,将Fluol Yellow 088溶解在乳酸中,并用胶带将其混合。D.溶解的荧光黄(左)和苯胺蓝(右)溶液。


设备


用于种植植物材料的生长室(Percival,CLF,Plant Climatics,AR75-L3)
涡旋混合器(Instrumentfirman Labor a,型号:Vortex-Genie 2)
水浴(Fisher Scientific)
不锈钢软镊子(3B DISSECTION SOFT TWEEZERS ,LABWORLD)
倒置共聚焦显微镜(Zeiss ,型号:LSM880)
注意:执行平铺扫描图像采集实验需要完全电动的X,Y,Z扫描台。

物镜:10 ×或20 × (适用于监视整个标本或根,例如,图3C)。对于更高分辨率的图像,需要更高的放大倍率,例如40 ×或63 × (图3D)
用于GFP和其他荧光报告基因的荧光信号检测系统(Shaner et al。,2007)

软件
共聚焦显微镜操作软件
ImageJ(Abràmoff等,2004)
Microsoft Excel
棱镜(GraphPad)

程序
样品染色
向每个装有2 ml荧光黄088(FY088)的孔中加入15-20株幼苗(例如,根据样品数量,为12孔板)(图2A)。
关闭盖子,并用封口膜密封板。将板放在70°C的铝箔筏上的水浴中20分钟(图2B-2D )。
注意:弯曲铝箔的边缘以防止下沉。在FY088溶液中孵育期间,请准备0.5%苯胺蓝溶液,并在室温下保持黑暗直至使用。

将幼苗从FY088解决方案转移到一个新的12孔板中,其中装有2 ml水,并在室温下孵育1分钟(图2E)。
通过移液除去水,然后向每个样品中加入2 ml 0.5%的苯胺蓝溶液(图2F)。孵育在20分钟内将在室温下黑暗。
在苯胺蓝温育后,将苗到一个新的- ,12-含2和24孔板- 3毫升水(图2G),孵育在10分钟的黑暗。

图2.样本染色。A.在2 ml FY088溶液中包含拟南芥幼苗的十二孔板。B.用石蜡膜密封的板。C.将密封好的孔板放在铝箔筏上。D.将样品置于70℃的水浴中20分钟。E.将幼苗在2毫升水中洗涤。F.含有2ml苯胺蓝溶液的孔板,带有幼苗。G.在2 ml水中洗涤苯胺蓝。H.准备带有石蜡膜条的显微镜载玻片,以制成“间隔物”。

样品制备/安装
切下两条封口膜,并在显微镜载玻片的两端缠绕2-3次(图2H和3A)。
在两个封口膜条之间的玻片中央添加几滴水(30-50μl )。使用镊子将幼苗放置在该液体上,并盖上盖玻片(图3A和3B)。
有关使用一个孔腔盖玻璃和定位苗的说明,请遵循协议先前描述:Marhavý和Benková ,(2015 )。

样品成像
准备倒置共焦显微镜以供使用;设置激光器(对于GFP:488 nm激发,发射500-550 nm)和物镜。
注意:要全面了解根中的suberin定位,我们建议使用10倍或20倍物镜(在干式或水浸模式下)执行平铺扫描成像。对于更高分辨率的图像,我们建议使用浸入水或油中的40 ×或63 ×物镜。

在Zeiss LSM 880上图像捕获的参数如图3C所示:图像大小 – x:21793.72 µm,y:18702.12 µm;缩放比例X – 1510 µm,缩放比例Y – 1510 µm;平铺扫描– tile1,重叠百分比:20.0,平铺模式:矩形网格(拼接图像);扫描模式–平面,缩放1.1;像素停留– 0.85 µs;主增益– 482;数字增益– 1.5;数字偏移量– 0.00;过滤器– SP 555;分束器– MBS:MBS 488 nm,MBS_InVis:平板,FW1:后部。

注意:应根据所用样品调整参数。

将载有苗的载玻片或培养皿安装在共聚焦显微镜的载物台上。
激活位置列表和瓷砖扫描(25个 ×与图像之间的20%的重叠34块)。
笔记:

根据大致扫描区域调整瓷砖数量。或者,激活自动缝合(也可以稍后使用共聚焦软件来完成)。
参数应根据样本的大小进行调整。
找到最靠近扫描区域中心点的根并标记位置。
停用位置并开始扫描。
注意小号:

使用上述参数,大约需要15分钟才能获得最终图像。如上所述,这提供了足够的时间来准备下一个样品。
如果实验室没有合适的共聚焦显微镜,则可以使用带平铺扫描选项的全电动荧光立体显微镜拍摄图像。
以TIF F或JPEG格式导出共焦图像。在ImageJ或其他软件中打开图像并分析您的图像(图3C,3D)。
注意:在每个步骤的时间安排和步骤上都必须准确,以确保结果的可重复性。





图3 。样品制备和成像。A. A 30 μ的升降水放置在两个封口膜条之间的显微镜载玻片上。B.在每张玻片上放10-15棵幼苗,并盖上盖玻片。C.对染色的拟南芥幼苗进行平铺扫描。黄色荧光表明木脂蛋白在根中沉积。白色框包围在D中展开的区域,以获得细木素薄片的详细图像。比例尺:1毫米。D.内胚层根细胞中的Suberin薄片的详细图像。比例尺:100μm。


数据分析


以Ima geJ Set比例打开图像;已知距离 长度单位(毫米或厘米);单击/标记全局,然后按确定。转到ImageJ,选择绘图线(分段或徒手画线),然后沿着根部从基部到尖端绘制一条线,按“ M”以测量根部长度。重复该过程以测量根部浸入区域的长度。将结果复制到Excel或Prisma GraphPad进行进一步处理(例如,计算根中的suberin占用率)。

注意:我们不建议您分析FY荧光强度,因为这可能会受到染色时间,洗涤和/或成像差异的影响。


菜谱


乙醇种子灭菌
将种子转移到2 ml Eppendorf试管中(每个试管中要灭菌的种子的最大量不得超过试管底部3 mm)

加入1 ml 70%乙醇,摇晃5秒钟,让种子沉降10分钟

在干净的长凳上干燥种子

半强度MS培养基(1升)
合并10克蔗糖,2.3克MS盐和0.5克MES,用KOH调节pH值至5.9,加入8克琼脂并用ddH 2 O填充至1升

高压灭菌介质20-30分钟,然后冷却至55 浇板前的°C

0.03%氟黄088溶液
在每次实验之前,必须新鲜制备室温下含乳酸的氟黄088(图1C,1D)。

注意:要加快将FY 088溶解在乳酸中,请将50 ml Falcon管连接到带有胶带的涡旋混合器上,并涡旋10-15分钟(图1C)。

0.5%苯胺蓝溶液
苯胺蓝在Milli-Q水

室温存放在阴暗处


致谢


我们AR (E T)^ h ankful来讨论尼科Geldner所有,并反馈给开发该协议。这项工作得到了瑞典研究委员会的PeterMarhavý的启动资助。沙希德·西迪克(Shahid Siddique)得到了美国农业部的资助(项目编号CA-D-ENM-2562-RR)。

该协议是基于Lux等人先前发表的工作改进的。(2005 )。


利益争夺


没有利益冲突或利益冲突。


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引用:Marhavy, P. and Siddique, S. (2021). Histochemical Staining of Suberin in Plant Roots. Bio-protocol 11(3): e3904. DOI: 10.21769/BioProtoc.3904.
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