搜索

Immunofluorescence Labeling of Pollen Tubes
花粉管的免疫荧光标记   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

Pollen tube is regarded as an excellent single-cell model system in plant cell studies. This protocol describes the use of a rapid and reliable immunofluorescence labeling method for studying in situ localization of proteins in pollen tubes. The whole experiment contains two major steps: pollen tube in vitro germination, and pollen tube fixation and immunolabeling. It takes about 2 days from pollen tube germination to immunofluorescence detection.

Materials and Reagents

  1. Lily, tobacco or Arabidopsis pollen tubes
    Note: Generally, mature lily and tobacco pollen grains are harvested 1-2 days after they are completely released from the anthers of flowers (Wang et al., 2010). For Arabidopsis, day-0 flowers are used for pollen collection (Boavida and McCormick, 2007).
  2. 1% glutaraldehyde
  3. 0.1% pectinase (Worthington Biochemical, catalog number: LS004298 )
  4. 1% cellulose (EMD Millipore, catalog number: 219466 )
  5. 1x phosphate buffered saline (PBS)
  6. 0.1% NaBH4 (INTERNATIONAL LABORATORY USA, catalog number: 1528397 )
  7. Primary antibody
  8. Secondary antibody
  9. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A-7906 )
  10. Sodium azide (Sigma-Aldrich, catalog number: S-2002 )
  11. NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I-8896 )
  12. Gelatin (Sigma-Aldrich, catalog number: 48723-F )
  13. Blocking buffer 1 (see Recipes)
  14. Blocking buffer 2 (see Recipes)
  15. Pollen species-specific germination media and culture conditions (see Recipes)

Equipment

  1. Centrifuge with free angle rotor (Eppendorf, model: 5810R )
  2. 0.22 μm syringe filter (Merck Millipore, catalog number: SLGV033NS )
  3. Confocal microscope (Leica Microsystems, model: TCS SP8 )
  4. Incubator
  5. Confocal dish (cover glass-bottom dish) (Life Scientific, catalog number: SPL-100350 )

Procedure

  1. In vitro pollen tube germination
    1. Lily, tobacco or Arabidopsis pollen tubes are in vitro germinated according to the methods described before (Wang and Jiang, 2011; Wang et al., 2013). In general, freshly harvest anthers from 1 lily (Lilium longiflorum) flower, 10-15 tobacco (Nicotiana tabacum) flowers or 30-40 Arabidopsis thaliana flowers. Transfer them into 20 ml species-specific pollen germination medium in a 50-ml Falcon tube. Vortex vigorously 1 min to release the pollen grains into the medium and remove the anthers with forceps. Spin down the pollen grains at 2,500 rpm for 5 min at room temperature (RT) using free angle rotor. Remove the supernatant and resuspend the pollen grains with species-specific pollen germination medium (5 ml for lily or tobacco pollens in a 50-ml conical tube and 250-300 μl for Arabidopsis pollens in a 2 ml Eppendorf tube).
      Pollen tube germination condition:
      Lily: 27.5 °C, 85 rpm/min shaker, 1.5-2.5 h;
      Tobacco: 27.5 °C, 85 rpm/min shaker, 2-3 h;
      Arabidopsis: 22.5 °C, water bath without shaking, 8-10 h.
    2. Spin down the germinated pollen tubes at 1,000 rpm for 5 min at RT using free angle rotor.

  2. Pollen tube fixation and immunolabeling
    1. Fix germinated pollen tubes with 1% glutaraldehyde in germination medium for 15 min at RT, 60 rpm/min shaker.
    2. Spin down the fixed pollen tubes at 1,000 rpm for 3 min at RT using free angle rotor. Sample washing procedure: Remove the supernatant and wash the pollen tubes by adding 2 ml pollen germination medium. Gently resuspend pollen tubes by mixing them in the medium by end-to-end rotating the tube by hands. Then keep the pollen tubes on bench undisturbed and wait for them naturally sink to the bottom of the tube at RT (repeat four times).
      Note: All the washing steps afterwards are performed as the same with step B2.
    3. Treat with 0.1% pectinase and 1% cellulase (prepared with 1x PBS) for 45 min at 28 °C (skip this step if immunolabel cell wall or cell wall associated proteins).
    4. Wash three times with 1x PBS.
    5. Transfer the pollen tubes to 10 ml of freshly prepared PBS containing 0.1% NaBH4 at 4 °C overnight without disturbance.
    6. Wash the cells twice by blocking buffer 1 (B1).
    7. Block the cells by B1 for 45 min at RT.
    8. Discard B1 and wash the cell with blocking buffer 2 (B2) once.
    9. Add primary antibody (in B2) and incubate at 4 °C overnight (1:50 or 1:100 dilution for polyclonal antibody serum or 4 μg/ml for affinity-purified antibody).
    10. Warm up the pollen tubes to RT and wash the cells with B2 twice. And then incubate pollen tubes with B2 for 30 min at RT without shaking.
    11. Remove supernatant B2, add secondary antibody (Alexa 1:3,000 dilution in B2) and keep the samples undisturbed in the dark for 1 h at RT.
    12. Wash the pollen tubes with B2 for three times and keep the samples in B2 in the dark without shaking (first time: 15 min, second time: 15 min, third time: 20 min).
    13. Add about 150 μl sample cells in confocal dish for confocal observation. Figure 1 shows the representative confocal images showing the results of immunofluorscences labeling of tobacco pollen tubes with anti-VSR antibodies (Figure 1A) and control labeling using anti-GFP antibodies (Figure 1B). The confocal fluorescence images were collected using a Leica TCS SP8 system with the following parameters: 63x water objective, 2x zoom, 900 gain, 0 background, 0.168 µm pixel size, photomultiplier tubes (PMTs) detector. The images from pollen tubes labeled with antibodies were collected with a laser level of ≤3% to ensure that the fluorescence signal was within the linear range of detection (typically 0.5 or 1% laser was used).


      Figure 1. Tobacco pollens were germinated for 2.5 h and subsequently fixed and immunolabeled with anti-VSR antibodies (A) and anti-GFP antibodies as a control (B). Scale bar = 25 µm. DIC: differential interference contrast

Recipes

  1. Blocking buffer 1
    1x PBS
    1% BSA
    Filter sterile, keep at 4 °C
  2. Blocking buffer 2
    1x PBS
    0.25% BSA
    0.25% gelatin
    0.05% NP-40
    0.02% azide
    Filter sterile and keep at 4 °C
  3. Pollen species-specific germination media and culture conditions
    SPECIES
    MEDIUM COMPOSITION
    Lily (Lilium longiflorum) pollen grains
    1.3 mM boric acid

    2.9 mM KNO3

    9.9 mM CaCl2

    10% (wt/vol) sucrose

    pH 5.8
    Tobacco (Nicotiana tabacum) pollen grains
    0.01% boric acid

    1 mM CaCl2

    1 mM Ca(NO3)2.4H2O

    1 mM MgSO4.7H2O

    10% (wt/vol) sucrose

    pH 6.5
    Arabidopsis thaliana pollen grains
    0.01% boric acid

    5 mM KCl

    1 mM MgSO4

    5 mM CaCl2

    10% (wt/vol) sucrose

    pH 7.5

Acknowledgments

The original version of this protocol was described in Wang et al. (2010). This work was supported by grants from the Research Grants Council of Hong Kong (CUHK 466011, 465112, 466613 and CUHK2/CRF/11G) to L.J.

References

  1. Boavida, L. C. and McCormick, S. (2007). TECHNICAL ADVANCE: Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana. Plant J 52(3): 570-582.
  2. Wang, H., Tse, Y. C., Law, A. H., Sun, S. S., Sun, Y. B., Xu, Z. F., Hillmer, S., Robinson, D. G. and Jiang, L. (2010). Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth. Plant J 61(5): 826-838.
  3. Wang, H. and Jiang, L. (2011). Transient expression and analysis of fluorescent reporter proteins in plant pollen tubes. Nat Protoc 6(4): 419-426.
  4. Wang, H., Zhuang, X., Cai, Y., Cheung, A. Y. and Jiang, L. (2013). Apical F-actin-regulated exocytic targeting of NtPPME1 is essential for construction and rigidity of the pollen tube cell wall. Plant J 76(3): 367-379.

简介

在植物细胞研究中花粉管被认为是一个优秀的单细胞模型系统。 该协议描述了使用快速和可靠的免疫荧光标记方法来研究原位蛋白质在花粉管中的定位。 整个实验包含两个主要步骤:花粉管在体外萌发,和花粉管固定和免疫标记。 从花粉管发芽到免疫荧光检测花费约2天。

材料和试剂

  1. 百合,烟草或拟南芥花粉管
    注意:通常,成熟百合和烟草花粉粒在它们从花的花药中完全释放后1-2天收获(Wang等,2010)。 对于拟南芥,将第0天的花用于花粉收集(Boavida和McCormick,2007)。
  2. 1%戊二醛
  3. 0.1%果胶酶(Worthington Biochemical,目录号:LS004298)
  4. 1%纤维素(EMD Millipore,目录号:219466)
  5. 1×磷酸盐缓冲盐水(PBS)
  6. 0.1%NaBH 4(INTERNATIONAL LABORATORY USA,目录号:1528397)
  7. 一抗
  8. 二抗
  9. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A-7906)
  10. 叠氮化钠(Sigma-Aldrich,目录号:S-2002)
  11. NP-40(IGEPAL CA-630)(Sigma-Aldrich,目录号:I-8896)
  12. 明胶(Sigma-Aldrich,目录号:48723-F)
  13. 阻止缓冲区1(请参阅配方)
  14. 阻止缓冲区2(参见配方)
  15. 花粉种特异性发芽培养基和培养条件(参见配方)

设备

  1. 使用自由角转子(Eppendorf,型号:5810R)离心,
  2. 0.22μm注射器过滤器(Merck Millipore,目录号:SLGV033NS)
  3. 共聚焦显微镜(Leica Microsystems,型号:TCS SP8)
  4. 孵化器
  5. 共焦盘(盖玻片 - 底部皿)(Life Scientific,目录号:SPL-100350)

程序

  1. 体外花粉管萌发
    1. 根据前面所述的方法,将百合,烟草或拟南芥花粉管体外萌发(Wang and Jiang,2011; Wang等人, 2013)。一般来说,从1个百合花(Lilium longiflorum)花,10-15个烟草( Nicotiana tabacum )花或30-40个拟南芥花的新鲜收获的花药 花。转移到20ml种子特异性花粉萌发培养基在50毫升Falcon管。涡旋大力1分钟释放花粉粒到介质中,用镊子除去花药。在室温(RT)下使用自由角转子以2,500rpm旋转花粉粒5分钟。除去上清液并用种特异性花粉萌发培养基(对于百合或烟草花粉在50ml锥形管中为5ml,对于拟南芥花粉为250-300μl,在2ml Eppendorf中重悬花粉颗粒管)。
      花粉管发芽条件:
      百合:27.5℃,85rpm/min振荡器,1.5-2.5h;
      烟草:27.5℃,85rpm/min振荡器,2-3小时;
      拟南芥:22.5℃,水浴不摇动,8-10小时。
    2. 在室温下使用自由角转子以1000rpm旋转发芽的花粉管5分钟。

  2. 花粉管固定和免疫标记
    1. 固定发芽的花粉管与1%戊二醛在萌发培养基中15分钟,在室温,60 rpm /分钟摇床。
    2. 在室温下使用自由角转子以1000rpm旋转固定花粉管3分钟。样品洗涤步骤:除去上清液,通过加入2ml花粉萌发培养基洗涤花粉管。轻轻地重悬花粉管通过将它们在介质中通过用手端对端旋转管而混合。然后保持花粉管在台上不受干扰,等待他们自然下沉到管底部RT(重复四次)。
      注意:之后的所有洗涤步骤与步骤B2相同。
    3. 用0.1%果胶酶和1%纤维素酶(用1×PBS制备)在28℃处理45分钟(如果免疫标记细胞壁或细胞壁相关蛋白,则跳过该步骤)。
    4. 用1x PBS洗涤三次。
    5. 在4℃下将花粉管转移到10ml含有0.1%NaBH 4的新鲜制备的PBS中过夜,无干扰。
    6. 用封闭缓冲液1(B1)洗涤细胞两次。
    7. 在室温下用B1封闭细胞45分钟。
    8. 弃去B1并用封闭缓冲液2(B2)洗涤细胞一次。
    9. 加入第一抗体(在B2中),并在4℃孵育过夜(对于多克隆抗体血清为1:50或1:100稀释,或对于亲和纯化的抗体为4μg/ml)。
    10. 将花粉管加热至RT,并用B2洗涤细胞两次。 然后在室温下用B2孵育花粉管30分钟,无振荡
    11. 除去上清液B2,加入二抗(Alexa 1:3000稀释在B2中),并保持样品在黑暗中在室温下1小时。
    12. 用B2洗涤花粉管三次,并在黑暗中将样品保持在黑暗中,不摇动(第一次:15分钟,第二次:15分钟,第三次:20分钟)。
    13. 在共聚焦皿中加入约150μl样品细胞用于共聚焦观察。图1显示了显示用抗VSR抗体(图1A)标记烟草花粉管的免疫荧光结果和使用抗GFP抗体的对照标记(图1B)的代表性共聚焦图像。使用具有以下参数的Leica TCS SP8系统收集共聚焦荧光图像:63x水物镜,2x变焦,900增益,0背景,0.168μm像素尺寸,光电倍增管(PMT)检测器。用≤3%的激光水平收集来自用抗体标记的花粉管的图像,以确保荧光信号在检测的线性范围内(通常使用0.5或1%激光)。


      图1.烟草花粉萌发2.5小时,随后固定并用抗VSR抗体(A)和抗GFP抗体作为对照(B)进行免疫标记。 比例尺= 25μm。 DIC:微分干涉对比度

食谱

  1. 阻塞缓冲区1
    1x PBS
    1%BSA
    过滤无菌,保持在4°C
  2. 阻塞缓冲区2
    1x PBS
    0.25%BSA 0.25%明胶
    0.05%NP-40
    0.02%叠氮化物 过滤无菌并保持在4°C
  3. 花粉种特异性发芽培养基和培养条件
    产品
    中等组分
    百合( 1.3mM硼酸

    2.9mM KNO 3

    9.9mM CaCl 2

    10%(wt/vol)蔗糖

    pH 5.8
    烟草( Nicotiana tabacum )花粉颗粒
    0.01%硼酸

    1mM CaCl 2

    1mM Ca(NO 3)2 Sub 2 O 4/4H 2 O 4 /

    1mM MgSO 4。7H 2 O 3

    10%(wt/vol)蔗糖

    pH 6.5
    拟南芥花粉粒
    0.01%硼酸

    5 mM KCl

    1mM MgSO 4

    5mM CaCl 2

    10%(wt/vol)蔗糖

    pH 7.5

致谢

该协议的原始版本在Wang等人(2010)中描述。这项工作得到香港研究资助局(CUHK 466011,465112,466613及CUHK2/CRF/11G)给予L.J.

参考文献

  1. Boavida,L.C。和McCormick,S。(2007)。 技术进步:温度作为增加和可再现性的决定性因素<
  2. Wang,H.,Tse,Y.C.,Law,A.H.,Sun,S.S.,Sun,Y.B.,Xu,Z.F.,Hillmer,S.,Robinson,D.G。和Jiang, 真空分选受体(VSR)和分泌性载体膜蛋白(SCAMPs )是花粉管生长所必需的。 61(5):826-838。
  3. Wang,H.和Jiang,L。(2011)。 植物花粉管中荧光报告蛋白的瞬时表达和分析。 Protoc 6(4): 419-426。
  4. Wang,H.,Zhuang,X.,Cai,Y.,Cheung,A.Y。和Jiang,L。(2013)。 NtPPME1的顶端F-肌动蛋白调节的胞吐靶向对于花粉管细胞的构建和刚性是必需的 。植物杂志76(3):367-379。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, H. and Jiang, L. (2014). Immunofluorescence Labeling of Pollen Tubes. Bio-protocol 4(10): e1131. DOI: 10.21769/BioProtoc.1131.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。