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[Bio101] Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis
[Bio101] 用启动子融合GUS的方法分析组织水平上基因的表达模式   

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Abstract

Promoter-driven GUS (beta-glucuronidase) activity is the most commonly used technique for tissue-specific expression patterns in Arabidopsis. In this procedure, GUS enzyme converts 5-bromo-4-chloro-3-indolyl glucuronide (X-Gluc) to a blue product. The staining is very sensitive. Processed samples can be examined under dissecting microscope or Differential Interference Contrast (Nomaski) microscope for bright blue color over cleared transparent background. Note this assay does not provide accurate information to subcellular levels.

Keywords: Gene expression(基因表达), GUS activity(GUS活性), Histostaining(免疫组化), Arabidopsis(拟南芥), Promoter activity(启动子活性)

Materials and Reagents

  1. Transgenic plants that contain genomic integration of a promoter: GUS expression cassette
  2. Potassium Ferrocyanide
  3. Potassium Ferricyanide
  4. Triton X-100
  5. 50 mM NaHPO4 buffer (pH 7.2)
  6. Dimethylformamide (DMF)
  7. Acetone
  8. NaHPO4 buffer
  9. 5-bromo-4-chloro-3-indolyl beta-D-glucuronide cyclohexamine salt (X-Gluc)
  10. 200 proof ethanol (once opened, 200 proof becomes essentially 190 proof)
  11. Staining buffer  (see Recipes)
  12. Stock solutions  (see Recipes)

Equipment

  1. Eppendorf tubes
  2. Vacuum
  3. Dissecting or light microscope
  4. Differential Interference Contrast (Nomaski) microscope

Procedure

  1. Harvest tissue and place in cold 90% Acetone on ice. This should stay on ice until all samples are harvested. For sample containers, Eppendorf tubes and glass scintillation vials work well.
  2. When all samples are harvested, place at room temperature (RT) for 20 min.
  3. Remove acetone from the samples, and add staining buffer on ice.
  4. Add X- Gluc to the staining buffer to a final concentration of 2 mM from a 100 mM stock solution of X-Gluc in DMF- this must be kept in the dark at -20 °C.
  5. Remove staining buffer from samples and add staining buffer with X-Gluc on ice.
    Note: Do not infiltrate when make LR embedding, instead infiltrate in the fixatives or 10% ethanol.
    Infiltrate the samples under vacuum, on ice, for 15 to 20 min. Release the vacuum slowly and verify that all the samples sink. If they don't, infiltrate again until they all sink to the bottom when the vacuum is released.
  6. Incubate at 37 °C (I usually do it for 2 h for strong promotors and up to overnight for weak promotors. It is not advisable from my experience to go too long (over two days) as the tissue seems to begin deteriorating during long incubations.
  7. Remove samples from incubator and remove staining buffer.
  8. Go through ethanol series from 10%, 30%, 50%, 70% (you may heat the sample to 60 °C to get rid of chloroplasts), to 95% (avoid light); 30 min each step and then finally 100%. You may store at 4 °C for up to a month, seal well.
  9. Go to embedding procedure, or observe directly under dissecting or light microscope. To mount, simply apply a few drops of water to the samples.

Recipes

  1. Staining buffer (final conc.) (fresh)
    0.2% Triton X-100 (may be reduced to 0.05%)
    50 mM NaHPO4 buffer (pH 7.2)
    2 mM potassium Ferrocyanide
    2 mM potassium Ferricyanide
    Water to volume
    Note: Higher Ferricyanide and ferrocyanide concentrations give lower overall staining level, but more specificity. 2 mM works well for most applications, but the concentrations may need to be adjusted for certain needs.
  2. Stock solutions (4 °C)
    10% Triton X-100
    0.5 M NaHPO4 buffer (pH 7.2)
    100 mM potassium Ferrocyanide (store in the dark at 4 °C)
    100 mM potassium Ferricyanide (store in the dark at 4 °C)
    100 mM X-Gluc in DMF

References

  1. Li, X., Chanroj, S., Wu, Z., Romanowsky, S. M., Harper, J. F. and Sze, H. (2008). A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process. Plant Physiol 147(4): 1675-1689.
  2. Padmanaban, S., Chanroj, S., Kwak, J. M., Li, X., Ward, J. M. and Sze, H. (2007). Participation of endomembrane cation/H+ exchanger AtCHX20 in osmoregulation of guard cells. Plant Physiol 144(1): 82-93.

简介

启动子驱动的GUS(β-葡糖醛酸糖苷酶)活性是拟南芥中组织特异性表达模式最常用的技术。 在该程序中,GUS酶将5-溴-4-氯-3-吲哚基葡糖苷酸(X-Gluc)转化为蓝色产物。 染色非常敏感。 处理的样品可以在解剖显微镜或微分干涉对比(Nomaski)显微镜下在透明背景上检查明亮的蓝色。 注意这个测定不提供准确的信息亚细胞水平。

关键字:基因表达, GUS活性, 免疫组化, 拟南芥, 启动子活性

材料和试剂

  1. 含有启动子:GUS表达盒的基因组整合的转基因植物
  2. 亚铁氰化钾
  3. 铁氰化钾
  4. Triton X-100
  5. 50mM NaHPO 4缓冲液(pH7.2)
  6. 二甲基甲酰胺(DMF)
  7. 丙酮
  8. NaHPO 4缓冲液
  9. 5-溴-4-氯-3-吲哚基β-D-葡萄糖醛酸环己胺盐(X-Gluc)
  10. 200 proof乙醇(一次打开,200 proof基本上190 proof)
  11. 染色缓冲区 (参见食谱)
  12. 股票解决方案 (见配方)

设备

  1. Eppendorf管
  2. 真空
  3. 解剖或光学显微镜
  4. 差分干涉对比(Nomaski)显微镜

程序

  1. 收获组织,并放置在冷的90%丙酮冰上。 这应该保持在冰上,直到所有的样品都收获。 对于样品容器,Eppendorf管和玻璃闪烁瓶工作良好。
  2. 当收获所有样品时,在室温(RT)下放置20分钟。
  3. 从样品中取出丙酮,在冰上加入染色缓冲液。
  4. 从染色缓冲液中加入X-Gluc至终浓度为2mM,来自100mM的X-Gluc在DMF中的储备溶液 - 这必须保存在-20℃的黑暗中。
  5. 从样品中取出染色缓冲液,在冰上加入X-Gluc染色缓冲液 注意:当使用LR包埋时,不要浸润,而是浸透固定剂或10%乙醇。
    在真空下,在冰上浸润样品15至20分钟。缓慢释放真空,并验证所有样品沉没。如果它们没有,则再次渗透,直到当真空释放时它们都沉到底部。
  6. 在37℃孵育(我通常对强启动子进行2小时,对弱启动子进行过夜,根据我的经验,不宜长时间(两天以上),因为组织在长时间孵育期间开始恶化。
  7. 从孵化器中取出样品,并去除染色缓冲液。
  8. 通过乙醇系列从10%,30%,50%,70%(你可以加热样品到60°C去除叶绿体),到95%(避免光);每步30分钟,然后最终100%。您可以在4°C储存长达一个月,密封好。
  9. 去嵌入程序,或直接在解剖或光学显微镜下观察。要安装,只需向样品滴几滴水。

食谱

  1. 染色缓冲液(最终浓度)(新鲜)
    0.2%Triton X-100(可以减少至0.05%) 50mM NaHPO 4缓冲液(pH 7.2) 2mM氰亚铁酸钾 2mM铁氰化钾 水量到
    注意:较高的铁氰化物和亚铁氰化物浓度提供较低的总染色水平,但更具特异性。 2 mM适用于大多数应用,但浓度可能需要根据某些需要进行调整。
  2. 储存溶液(4℃)
    10%Triton X-100 0.5M NaHPO 4缓冲液(pH 7.2) 100mM氰亚铁酸钾(在4℃黑暗中储存) 100mM铁氰化钾(储存在4℃的黑暗中) 100mM X-Gluc的DMF溶液

参考文献

  1. Li,X.,Chanroj,S.,Wu,Z.,Romanowsky,S.M.,Harper,J.F。和Sze,H。(2008)。 不同的内体Ca 2 + /Mn 2+ 泵通过分泌过程影响根生长。 植物生理 147(4):1675-1689。
  2. Padmanaban,S.,Chanroj,S.,Kwak,J.M.,Li,X.,Ward,J.M。和Sze,H。(2007)。 内膜阳离子/H + 交换剂AtCHX20参与保卫细胞的渗透调节 。 Plant Physiol 144(1):82-93。
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, X. (2011). Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis. Bio-protocol Bio101: e93. DOI: 10.21769/BioProtoc.93;
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our leaf can't stain,however the stain solution become blue .Do you know the reason?please
12/1/2014 5:24:47 PM Reply
Dieu Linh Nguyen Trieu
University of Science and Technology of Hanoi
For the first step, why we have to place in cold 90% Acetone on ice, (why Acetone, why ice?)
Thank you so much!
Please answer as much details as possible, I'm just a 3rd year of Bachelor :)
Thank again
6/20/2014 12:56:43 AM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Cold acetone is for fixation, a way to preserve the cell/tissue structures as in vivo.

6/23/2014 2:01:30 PM


youtao chen
Beijing Omeage Bio-tech Co.,LTD
9/14/2011 11:45:23 PM Reply
Xiyan Li
Department of Genetics, Stanford University, USA

Since cell lysis is not involved in your experiment, you can use your sample as cell lysate in most assays developed for cells. Very general methods can also be found in literature or on the web, and commercial fluorescence-based reagents from several vendors such as Promega, Pierce, and others. Search for "beta-galactosidase fluorescence" will yield enough information.

9/16/2011 5:40:58 AM


Yuanqing Lin
What the function of Potasium fero and feri cyanide in gus substrate?
7/8/2011 2:55:05 PM Reply
bio-protocol

Ferro- and Ferri-cyanides form a system buffering redox status (mechanistically like pH buffer). The hydrolyzed indolyl half of X-Gluc by GUS needs to be oxidized to radical before it can form the dimerized blue precipitate as so-called GUS staining.

7/12/2011 1:06:39 AM