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Confocal laser scanning microscopy in combination with fluorescent proteins is a powerful tool for the study of sexual reproduction and other developmental processes in plants. In order to understand the origin and localization of fluorescent signals in a complex tissue, staining of cell outlines is often mandatory. Cell wall staining with SCRI Renaissance 2200 (SR2200) has recently been described as a method of choice to study plant reproductive processes (Musielak et al., 2015). In this protocol, we present detailed instructions on the use of SR2200 to stain cell walls in different Arabidopsis tissues.

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Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots
SCRI Renaissance 2200 (SR2200)试剂作为通用染料可用于发育胚胎、完整胚珠、花粉管和根部的成像

植物科学 > 植物细胞生物学 > 细胞染色
作者: Thomas J. Musielak
Thomas J. MusielakAffiliation: Max Planck Institute for Developmental Biology, Department of Cell Biology, Tuebingen, Germany
Bio-protocol author page: a3518
Patrick Bürgel
Patrick BürgelAffiliation: Max Planck Institute for Developmental Biology, Department of Cell Biology, Tuebingen, Germany
Bio-protocol author page: a3519
Martina Kolb
Martina KolbAffiliation: Max Planck Institute for Developmental Biology, Department of Cell Biology, Tuebingen, Germany
Bio-protocol author page: a3520
 and Martin Bayer
Martin BayerAffiliation: Max Planck Institute for Developmental Biology, Department of Cell Biology, Tuebingen, Germany
For correspondence: martin.bayer@tuebingen.mpg.de
Bio-protocol author page: a3521
Vol 6, Iss 18, 9/20/2016, 1587 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1935

[Abstract] Confocal laser scanning microscopy in combination with fluorescent proteins is a powerful tool for the study of sexual reproduction and other developmental processes in plants. In order to understand the origin and localization of fluorescent signals in a complex tissue, staining of cell outlines is often mandatory. Cell wall staining with SCRI Renaissance 2200 (SR2200) has recently been described as a method of choice to study plant reproductive processes (Musielak et al., 2015). In this protocol, we present detailed instructions on the use of SR2200 to stain cell walls in different Arabidopsis tissues.
Keywords: Renaissance SR2200(文艺复兴时期的sr2200), Cell wall staining(细胞壁染色), Arabidopsis thaliana(拟南芥), Pollen tube(花粉管), Confocal microscopy(共聚焦显微镜)

[Abstract]

Materials and Reagents

  1. Liquid blocker, “PAP pen” (Science Services, catalog number: N71310 )
  2. Microscope slides (e.g., Superfrost; Carl Roth, catalog number: 1879.1 )
  3. Double-sided adhesive tape (e.g., reichelt elektronik, Tesa Sande, catalog number: 0 5338 )
  4. Syringe needle (e.g., Terumo, catalog number: NN-2425R ; VWR International, catalog number: HSWA4710005525 )
  5. Microscope cover slips (e.g., Carl Roth, catalog number: H874.2 )
  6. Pipette tips (200 μl)
  7. Razor blade (e.g., VWR International, Darmstadt, catalog number: 233-5224 )
  8. Fresh Arabidopsis plant material (growth conditions as described in Babu et al., 2013)
  9. Deionized water
  10. 10% glycerol (e.g., Sigma-Aldrich, catalog number: 49782 )
  11. 1% (v/v) DMSO (e.g., Carl Roth, catalog number: 7029.2 )
  12. 0.05% (w/v) Triton-X 100 (e.g., Carl Roth, catalog number: 3051.2 )
  13. 3.75% (w/v) para-formaldehyde (e.g., Carl Roth, catalog number: 0 335.2 )
  14. PBS buffer (pH 7.4) (e.g., Carl Roth, catalog number: 9143.2 )
  15. Optional: 4’,6-diamidin-2-phenylindol (DAPI) (e.g., Carl Roth, catalog number: 6335.1 )
  16. SR2200 staining solution (see Recipes)
    Note: SR2200 can be ordered from: Renaissance Chemicals Ltd, Unit 1 Blackwood Hall Business Park, North Duffield, Selby, UK. Contact: Howard Weaver, enquiries@renchem.co.uk.

Equipment

  1. Tweezers
  2. Binocular (e.g., ZEISS, model: Stemi 2000 )
  3. Adjustable pipette 20-200 μl (e.g., Gilson Pipetman-neo P200N; Carl Roth, catalog number: HY87.1 )
  4. Vacuum pump (e.g., Vacuubrand PC2004 Vario)
  5. Vacuum chamber (e.g., Kartell Labware 554)
  6. Confocal laser scanning microscope with 405 nm laser and standard filter set for DAPI imaging (e.g., Leica TCS SP8 ) or spectral detector if double staining with DAPI is required (e.g., Zeiss LSM780NLO with 32 channel GaAsP array)

Procedure

  1. Preparation and staining of Arabidopsis embryos (Figure 1)
    1. Using the PAP pen, mark a square on a microscope slide that frames a 20 mm by 20 mm area.
    2. Add 60 μl Renaissance staining solution to the center of the marked square.
    3. Remove a silique of the appropriate developmental stage with tweezers and transfer to double-sided tape on a microscope slide.
    4. Steps A5-8 will be carried out under a stereo microscope/binocular.
    5. Cut the silique open alongside the septum with a syringe needle (Video 1).

      Video 1. Dissecting siliques to collect ovules

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    6. Gently pull the carpels to the side.
    7. Ovules can be picked up with the syringe needle tip and transferred to the staining solution on the microscope slide.
    8. Carefully cover the ovules with a cover slip avoiding air bubbles.
    9. Squeeze out the embryos from the seed by applying pressure on the cover slip with a pipette tip until ovules burst open. Slowly release the pressure to avoid turbulences that could break off the suspensor structure from the embryo (Video 2).
    10. Embryos can immediately be imaged by confocal microscopy.

      Video 2. Releasing embryos from dissected ovules by applying pressure to the coverslip

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player



      Figure 1. Preparation and staining of Arabidopsis embryos. A. Using the PAP pen, a 20 x 20 mm square is marked on a microscope slide. B. The square is filled with 60 µl SR2200 staining solution. C. A silique of the appropriate developmental stage is transferred to double-sided tape on a microscope slide. D. The silique is cut open using the tip of a syringe needle. E. Ovules are collected and transferred to the staining solution. F. After carefully covering the ovules with a cover slip, the embryos are squeezed out of the ovules by gently applying pressure to the cover slip with a pipet tip.

  2. Preparation and staining of young ovules and pollen tubes
    1. Follow steps A1-7 of embryo preparation protocol.
    2. Unfertilized or freshly fertilized ovules (up to 2 days after pollination) as well as whole silique tissue can be transferred to the staining solution on the microscope slide. For young ovules, it is easiest to transfer the whole replum and septum with attached ovules.
    3. Carefully transfer the microscope slide to a vacuum chamber and apply soft vacuum (80 mbar) for 5 min.
    4. Remove the SR2200 staining solution with a pipette without picking up the stained plant tissue.
    5. Add 150 µl water.
    6. Apply soft vacuum (80 mbar) for 5 min.
    7. Carefully remove the water with a pipette without picking up the plant tissue.
    8. Add 50 μl 10% glycerol solution.
    9. Gently cover the sample with a cover slip without trapping air bubbles.
    10. The sample can immediately be used for confocal microscopy.

  3. Preparation and staining of root tissue (Figure 2)


    Figure 2. Preparation and staining of Arabidopsis roots. A. 5-day-old seedlings are placed on a prepared microscope slide and cut at the hypocotyl with a scalpel knife or razor blade before staining with SR2200 solution; B. pWOX5::erGFP expression (green) (Xu et al., 2006) in the quiescent center cells. SR2200 staining in grey. Scale bar = 50 µm.

    1. Using the PAP pen, mark a square on a microscope slide that frames a 20 mm by 20 mm area.
    2. Transfer 4-5 seedlings to the object slide and position them within the square so that the roots are all orientated in one direction.
    3. Take a razor blade and cut horizontally below the hypocotyl.
    4. Add 100 μl SR2200 staining solution.
    5. Apply soft vacuum (80 mbar) for 5 min.
    6. Carefully remove the staining solution with a pipette without touching the roots.
    7. Add 100 µl water.
    8. Apply soft vacuum (80 mbar) for 5 min.
    9. Carefully remove the water with a pipette without touching the roots.
    10. Add 50 μl 10% glycerol solution.
    11. Gently cover the sample with a cover slip.
    12. The sample can immediately be used for confocal microscopy.

  4. Microscopy
    SR2200 has an excitation maximum around 350 nm but a 405 nm laser line can be used successfully. SR2200 gives a very bright fluorescence signal, therefore only very limited laser power is required. Emission can be detected using commonly available filter sets designed for DAPI imaging.
    When double-staining with SR2200 and DAPI is desired, simultaneous imaging of both dyes in a single channel usually leads to a very weak DAPI signal in comparison to the much brighter SR2200 fluorescence. Therefore it is a more suitable approach to record spectral information of the fluorescence signal, for example with a detector array, and separate the signal into individual channels by spectral unmixing. We used a Zeiss LSM780 microscope with a 32-channel detector array in combination with the online fingerprinting function of the ZEN software and applied individually pre-recorded spectra of the two dyes as well as background fluorescence. The signal of the DAPI channel was increased around 18-fold in comparison to the signal of SR2200 by adjusting the digital gain of the individual channels to get comparable signal intensities. The staining procedure is as described above with the addition of DAPI to the staining solution.

Representative data



Figure 3. Representative images of SR2200-stained Arabidopsis embryos. A. 8-cell embryo expressing pNMA>>NLS-tdtomato (red) (Babu et al., 2013). B. Late heart-stage embryo expressing Q0990>>erGFP (green) (Weijers et al., 2006). SR2200 signal in grey. C. Maximum projection of 2-cell embryo double-stained with SR2200 and DAPI. DAPI signal in nuclei (cyan) was separated from SR2200 signal in cell walls (grey) by spectral unmixing (Musielak et al., 2015). Scale bar = 20 µm.

Recipes

  1. SR2200 staining solution
    Note: Stock solution of the supplier is considered as 100%.
    0.1% (v/v) SR2200
    1% (v/v) DMSO
    0.05% (w/v) Triton-X 100
    5% (w/v) glycerol
    3.75% (w/v) para-formaldehyde
    in PBS buffer (pH 7.4)
    Optional: 2 µg/ml DAPI
    To simplify the preparation, we first prepare a 4% (w/v) para-formaldehyde (PFA) solution by dissolving the PFA powder in gently heated 1x PBS buffer (pH 7.4). After cooling of the PFA solution, we add the rest of the ingredients to make up the final volume. The staining solution can be stored in 1 ml aliquots at -20 °C. Always use fresh aliquots for staining.

Acknowledgments

This protocol was adapted from the previously published study, Musielak et al. (2015). We would like to thank Christian Liebig and Aurora Panzera of the microscope facility at the MPI for Developmental Biology for technical assistance and support, Agnes Henschen and Laura Schenkel for their help in establishing the initial staining protocol. Research in our group is supported by the German Science Foundation (Deutsche Forschungsgemeinschaft-DFG: SFB1101/B01 to M.B.) and the Max Planck Society.

References

  1. Babu, Y., Musielak, T., Henschen, A. and Bayer, M. (2013). Suspensor length determines developmental progression of the embryo in Arabidopsis. Plant Physiol 162(3): 1448-1458.
  2. Musielak, T. J., Schenkel, L., Kolb, M., Henschen, A. and Bayer, M. (2015). A simple and versatile cell wall staining protocol to study plant reproduction. Plant Reprod 28(3-4): 161-169.
  3. Weijers, D., Schlereth, A., Ehrismann, J. S., Schwank, G., Kientz, M. and Jurgens, G. (2006). Auxin triggers transient local signaling for cell specification in Arabidopsis embryogenesis. Dev Cell 10(2): 265-270.
  4. Xu, J., Hofhuis, H., Heidstra, R., Sauer, M., Friml, J. and Scheres, B. (2006). A molecular framework for plant regeneration. Science 311(5759): 385-388.

材料和试剂

  1. 液体阻滞剂,"PAP pen"(Science Services,目录号:N71310)
  2. 显微镜载玻片(例如,Superfrost; Carl Roth,目录号:1879.1)
  3. 双面胶带(例如,reichelt elektronik,Tesa Sande,目录号:05338)
  4. 注射器针(例如,Terumo,目录号:NN-2425R; VWR International,目录号:HSWA4710005525)
  5. 显微镜盖玻片(,例如,Carl Roth,目录号:H874.2)
  6. 移液管吸头(200μl)
  7. 剃刀刀片(,VWR International,Darmstadt,目录号:233-5224)
  8. 新鲜拟南芥植物材料(生长条件如Babu等人在2013年描述的)。</b>
  9. 去离子水
  10. 10%甘油(例如,Sigma-Aldrich,目录号:49782)
  11. 1%(v/v)DMSO(例如,Carl Roth,目录号:7029.2)
  12. 0.05%(w/v)Triton-X 100(例如,Carl Roth,目录号:3051.2)
  13. 3.75%(w/v)多聚甲醛(例如,Carl Roth,目录号:0335.2)
  14. PBS缓冲液(pH 7.4)(例如,Carl Roth,目录号:9143.2)
  15. 任选的:4',6-二脒基-2-苯基吲哚(DAPI)(例如,Carl Roth,目录号:6335.1)
  16. SR2200染色溶液(见配方)
    注意:SR2200可订购自:Renaissance Chemicals Ltd,Unit 1 Blackwood Hall Business Park,North Duffield,Selby,UK。 联系人:Howard Weaver, enquiries@renchem.co.uk /a> 。

设备

  1. 镊子
  2. 双目(例如,ZEISS,型号:Stemi 2000)
  3. 可调移液管20-200μl(例如,Gilson Pipetman-neo P200N; Carl Roth,目录号:HY87.1)
  4. 真空泵(,例如,Vacuubrand PC2004 Vario)
  5. 真空室(例如,Kartell Labware 554)
  6. 具有用于DAPI成像(例如,Leica TCS SP8)的405nm激光和标准滤光片组的共聚焦激光扫描显微镜或如果需要使用DAPI双重染色的光谱检测器(例如。 ,Zeiss LSM780NLO 与32通道GaAsP阵列)

程序

  1. 拟南芥胚胎的制备和染色(图1)
    1. 使用PAP笔,在构成20mm×20mm面积的显微镜载玻片上标记一个正方形。
    2. 向标记的正方形的中心加入60μl文艺复兴染色溶液
    3. 用镊子取出适当发育阶段的长角果,并转移到显微镜载玻片上的双面胶带。
    4. 步骤A5-8将在立体显微镜/双目镜下进行。
    5. 用注射器针头(视频1)沿着隔膜切开长角果。

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    6. 轻轻地将心皮拉向侧边。
    7. 可以用注射器针尖取出胚珠,并转移到显微镜载玻片上的染色溶液中
    8. 小心地覆盖胚珠,盖玻片避免气泡。
    9. 通过用移液管尖端在盖玻片上施加压力挤出胚芽从种子挤出,直到胚珠破裂。缓慢释放压力,以避免可能从胚胎中断悬吊器结构的湍流(视频2)。
    10. 胚胎可以立即通过共聚焦显微镜成像
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      图1.拟南芥胚胎的制备和染色。A.使用PAP笔,在显微镜载玻片上标记20×20mm正方形。 B.用60μlSR2200染色溶液填充正方形。 C.将适当发育阶段的长角果转移到显微镜载玻片上的双面胶带上。 D.使用注射器针头的尖端将长角果切开。 E.收集胚珠并转移到染色溶液中。 F.用盖玻片小心地覆盖胚珠后,通过用移液管尖端轻轻地向盖玻片施加压力将胚胎挤出胚珠。

  2. 年轻胚珠和花粉管的制备和染色
    1. 按照胚胎制备方案的步骤A1-7。
    2. 未受精或新受精的胚珠(授粉后至多2天)以及整个长角果组织可以转移到显微镜载玻片上的染色溶液中。 对于年轻的胚珠,最容易将整个复制品和隔膜与附着的胚珠一起转移
    3. 小心地将显微镜载片转移到真空室,并施加软真空(80毫巴)5分钟
    4. 用移液管除去SR2200染色溶液,但不摘取染色的植物组织
    5. 加入150μl水。
    6. 使用软真空(80 mbar)5分钟。
    7. 用移液管小心取出水,不要取出植物组织。
    8. 加入50μl10%甘油溶液
    9. 用盖玻片轻轻盖住样品,不要夹住气泡。
    10. 样品可立即用于共聚焦显微镜
  3. 根组织的制备和染色(图2)


    图2.拟南芥根的制备和染色。将5天龄的幼苗放置在准备的显微镜载玻片上,用手术刀或剃刀在下胚轴上切割刀片用SR2200溶液染色; b。在静止中心细胞中的pWOX5 :: erGFP 表达(绿色)(Xu等人,2006)。 SR2200染色为灰色。比例尺=50μm。

    1. 使用PAP笔,在构成20mm×20mm面积的显微镜载玻片上标记一个正方形。
    2. 将4-5个幼苗转移到物体上,并将它们放置在正方形内,使得根部都在一个方向上定向
    3. 拿起剃刀刀片,水平切割下胚轴。
    4. 加入100μlSR2200染色溶液
    5. 使用软真空(80 mbar)5分钟。
    6. 小心地用移液管除去染色溶液而不接触根
    7. 加入100μl水。
    8. 使用软真空(80 mbar)5分钟。
    9. 小心地用移液管移除水,而不接触根
    10. 加入50μl10%甘油溶液
    11. 用盖玻片轻轻盖住样品。
    12. 样品可立即用于共聚焦显微镜
  4. 显微镜
    SR2200具有350 nm左右的激发最大值,但可以成功使用405 nm激光线。 SR2200给出非常明亮的荧光信号,因此只需要非常有限的激光功率。可以使用为DAPI成像设计的通常可用的过滤器组来检测发射。
    当需要用SR2200和DAPI双染色时,与更明亮的SR2200荧光相比,在单个通道中同时成像两种染料通常导致非常弱的DAPI信号。因此,它是更合适的方法,例如用检测器阵列记录荧光信号的光谱信息,并通过光谱解混合将信号分离成单个通道。我们使用具有32通道检测器阵列的Zeiss LSM780显微镜与ZEN软件的在线指纹功能组合,并且应用单独预先记录的两种染料的光谱以及背景荧光。与SR2200的信号相比,通过调整各个通道的数字增益以获得可比较的信号强度,DAPI通道的信号增加约18倍。染色程序如上所述,在染色溶液中加入DAPI

代表数据



图3.SR2200染色的拟南芥胚胎的代表性图像表达pNMA> NLS-tdtomato(红色)的A.8-细胞胚胎 (Babu等人,2013)。 B.表达Q0990> erGFP(绿色)的晚期心脏期胚胎(Weijers等人,2006)。 SR2200信号为灰色。 C.用SR2200和DAPI双染色的2细胞胚胎的最大投影。 通过光谱解混合(Musielak等人,2015),将细胞核中的DAPI信号(青色)与细胞壁中的SR2200信号(灰色)分离。 比例尺=20μm。

食谱

  1. SR2200染色溶液
    注意:供应商的库存解决方案被视为100%。
    0.1%(v/v)SR2200
    1%(v/v)DMSO 0.05%(w/v)Triton-X 100
    5%(w/v)甘油 3.75%(w/v)多聚甲醛
    在PBS缓冲液(pH7.4)中 可选:2μg/ml DAPI
    为了简化制备,我们首先通过将PFA粉末溶解在温和加热的1×PBS缓冲液(pH 7.4)中来制备4%(w/v)多聚甲醛(PFA)溶液。冷却PFA溶液后,我们加入剩余的成分以补足最终体积。染色溶液可以在-20℃下以1ml等分试样储存。始终使用新鲜等分试样进行染色。

致谢

该方案改编自以前发表的Musielak等人的研究 。 (2015)。我们要感谢Christian Liebig和Aurora Panzera的发展生物学MPI的显微镜设备的技术援助和支持,Agnes Henschen和Laura Schenkel帮助建立初始染色方案。我们集团的研究由德国科学基金会(Deutsche Forschungsgemeinschaft-DFG:SFB1101/B01至M.B.)和马克斯普朗克学会支持。

参考文献

  1. Babu,Y.,Musielak,T.,Henschen,A.和Bayer,M。(2013)。  悬浮体长度决定胚胎在拟南芥中的发育进程。 162(3):1448-1458 。
  2. Musielak,TJ,Schenkel,L.,Kolb,M.,Henschen,A.和Bayer,M.(2015)。  研究植物繁殖的简单而多功能的细胞壁染色方案。植物繁殖 28(3-4)
  3. Weijers,D.,Schlereth,A.,Ehrismann,JS,Schwank,G.,Kientz,M。和Jurgens,G.(2006)。  生长素在拟南芥胚胎发生中触发细胞规格的瞬时局部信号传导。 Dev Cell em> 10(2):265-270
  4. Xu,J.,Hofhuis,H.,Heidstra,R.,Sauer,M.,Friml,J。和Scheres,B.(2006)。  用于植物再生的分子框架。 311(5759):385-388。
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How to cite this protocol: Musielak, T. J., Bürgel, P., Kolb, M. and Bayer, M. (2016). Use of SCRI Renaissance 2200 (SR2200) as a Versatile Dye for Imaging of Developing Embryos, Whole Ovules, Pollen Tubes and Roots. Bio-protocol 6(18): e1935. DOI: 10.21769/BioProtoc.1935; Full Text



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我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

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  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


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