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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, 15282 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).

 

 

方法和试剂

 

母液

1.       冰醋酸

2.       乙醇

3.       1M NaOH (氢氧化钠)

4.       100ml 1M K2HPO4 (磷酸氢二钾)

5.       100ml 1M KH2PO4 (磷酸二氢钾)

6.       苯胺蓝 (Fisher)

7.       甘油

工作溶液

1.       冰醋酸含量为10% 的乙醇溶液 (固定组织使用)

2.       50 mM 偏磷酸钾溶液: 4.17ml 1M 磷酸氢二钾 + 0.83 ml 1M 磷酸二氢钾 + 995 ml, pH 7.5

3.       苯胺蓝含量为0.01% KPO4 溶液 (染色)

4.       11.含有50%甘油的偏磷酸钾溶液(封固剂)

 

仪器

 

1.       紫外显微镜

 

步骤

 

1.       1.5ml离心管里加入250μl冰醋酸,将雄蕊浸泡到冰醋酸里至少固定1.5小时。如果需要可将组织过夜固定。

2.       将固定完的组织浸泡在1M氢氧化钠溶液里过夜处理,是组织软化。

3.       用偏磷酸钾洗植物组织三次.  (在这个阶段植物组织是易碎的)

4.       200μl苯胺蓝对组织进行染色5-10分钟或者最久可以染色10小时。

5.       染色后,将组织放置到载玻片上在紫外显微镜下观察。如有需要可以压片观察。

 

参考文献

 

1.        Modified from the online protocol on Dr. Daphne Preusss’s lab (Univ of Chicago).

2.        Lu Y., Chanroj S., Zulkifli L., Johnson M.A., Uozumi N., Cheung A., Sze H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93. 

 

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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  

Anonymous 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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