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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.

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[Bio101] Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis

Cell Biology > Tissue analysis > Tissue staining
Author: Xiyan Li
Xiyan LiAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: lixiyan@stanford.edu
Bio-protocol author page: a13
7/5/2011, 14629 views, 4 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.93

[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, GUS activity, Histostaining, Arabidopsis, Promoter activity

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.


How to cite this protocol: Li, X. (2011). Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis. Bio-protocol Bio101: e93. DOI: 10.21769/BioProtoc.93; Full Text



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12/1/2014 5:24:47 PM  

our leaf can't stain,however the stain solution become blue .Do you know the reason?please

Reply

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6/20/2014 12:56:43 AM  

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

6/23/2014 2:01:30 PM  

Xiyan Li (Author)
Department of Genetics,Stanford University

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

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9/14/2011 11:45:23 PM  

youtao chen
Beijing Omeage Bio-tech Co.,LTD

Dear Xiyan:

您好,请问GUS基因在体外表达,怎么检测呢,有这方面的protocol吗?谢谢!

9/16/2011 5:40:58 AM  

Xiyan Li (Author)
Department of Genetics,Stanford University

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.

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7/8/2011 2:55:05 PM  

Anonymous Lin
Test institute

What the function of Potasium fero and feri cyanide in gus substrate?

7/12/2011 1:06:39 AM  

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.

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