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The aim of this experiment is to track pollen tube growth in vivo in the female tissues after pollination. This can be used to phenotype pollen germination, tube growth and guidance, and reception.

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[Bio101] Arabidopsis Pollen Tube Aniline Blue Staining
[Bio101] 苯胺蓝染色法研究拟南芥花粉管的生长发育

细胞生物学 > 组织分析 > 组织染色
作者: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Scienc, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
6/20/2011, 16040 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.88

[Abstract] The aim of this experiment is to track pollen tube growth in vivo in the female tissues after pollination. This can be used to phenotype pollen germination, tube growth and guidance, and reception.

[Abstract]

Materials and Reagents

  1. Stock solutions:
    1. Acetic acid
    2. Ethanol
    3. NaOH
    4. K2HPO4
    5. KH2PO4
    6. Aniline blue (Thermo Fisher Scientific)
    7. Glycerol
  2. Working solutions:
    1. 10% acetic acid in EtOH (fixative)
    2. 0.01% aniline blue in KPO4 buffer (dye)
    3. KPO4 buffer made with 50% glycerol (mounting media)
    4. KPO4 buffer (see Recipes)

Equipment

  1. Microscope with UV light

Procedure

  1. Submerge pistil tissue in 250 µl acetic acid and fix it for 1.5 h or more in an Eppendorf tube. Tissue can be fixed overnight if necessary.
  2. Soften tissue by submerging it in 1 M NaOH overnight.
  3. Wash 3 times with KPO4 buffer (tissue is fragile at this stage).
  4. Stain with 200 µl aniline blue for 5-10 min or as long as 10 h.
  5. Transfer to a slide, add mounting media and observe under UV. Squash if necessary.

Recipes

  1. 50 mM KPO4 buffer (pH 7.5)
    4.17 ml 1 M K2HPO4
    0.83 ml 1 M KH2PO4
    995 ml H2O

References

  1. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.
  2. Modified from the online protocol on Dr. Daphne Preusss’s lab (Univ of Chicago).

材料和试剂

  1. 库存解决方案:
    1. 醋酸
    2. 乙醇
    3. NaOH
    4. K 2 HPO 4
    5. KH sub 2 PO 4
    6. 苯胺蓝(Thermo Fisher Scientific)
    7. Glycerol
  2. 工作解决方案:
    1. 10%乙酸的EtOH溶液(固定剂)
    2. 在KPO 4缓冲液(染料)中的0.01%苯胺蓝,
    3. 用50%甘油制备的KPO 4缓冲液(封固介质)
    4. KPO 4 缓冲区(请参阅配方)

设备

  1. 显微镜与紫外光

程序

  1. 浸没雌蕊组织在250微升醋酸中,并将其固定在Eppendorf管中1.5小时或更长时间。 如果需要,组织可以固定过夜。
  2. 通过将其浸没在1M NaOH中过夜来软化组织。
  3. 用KPO 4缓冲液洗涤3次(在该阶段组织是脆弱的)。
  4. 用200μl苯胺蓝染色5-10分钟或长达10小时。
  5. 转移到载玻片,添加安装介质,在紫外线下观察。 必要时加入壁球。

食谱

  1. 50mM KPO 4缓冲液(pH7.5) 4.17ml 1M K 2 HPO 4
    0.83ml 1M KH 2 PO 4>/
    995ml H 2 O·dm /

参考文献

  1. Lu,Y.,Chanroj,S.,Zulkifli,L.,Johnson,M.A.,Uozumi,N.,Cheung,A.and Sze,H。(2011)。 缺少一对K +转运蛋白的花粉管未能在拟南芥中靶向胚珠 。 Plant Cell 23(1):81-93。
  2. 从Daphne Preusss博士实验室(芝加哥大学)的在线协议修改。
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How to cite this protocol: Lu, Y. (2011). Arabidopsis Pollen Tube Aniline Blue Staining. Bio-protocol Bio101: e88. DOI: 10.21769/BioProtoc.88; Full Text



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3/26/2015 6:02:52 AM  

”4.17ml 1M 磷酸氢二钾 + 0.83 ml 1M 磷酸二氢钾 + 995 ml水“这是5mM的吧?

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3/10/2015 12:17:09 AM  

苯胺蓝染色的原理是什么?与固绿染色原理有什么区别?

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4/22/2012 12:06:39 AM  


hi , can i use this method for observation of pollen tube growth "in embryo sac" ?

thanks

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7/11/2011 2:20:50 PM  

Yuanqing Lin
Test institute

苯胺蓝染色花粉能够显示花粉什么指标:花粉内含物?活力?核?与 Alexander,DAPI,FDA染色花粉等有什么区别

7/11/2011 2:21:17 PM  

bio-protocol

Aniline blue stains callose, which is a major chemical component of the pollen tube cell wall. That makes it a good dye to visualize pollen tube growing in vivo.

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