Claudia Castillo-González
  • Post-Doc, Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX
Research fields
  • Plant Science
Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis
Author:  Xiyan Li, date: 07/05/2011, view: 31316, Q&A: 3
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.
It worked but I had to optimize it with several modifications.
[Feedback 1] The protocol was very helpful. I ended up using a few modifications for the conditions in my lab. Specifically, I dissolved the X-Gluc in Methanol, not in DMF. I prepared a 20mM stock and I used a final concentration of 2mM instead of 1mM, this was likely necessary because the reagents in my lab might have been too old. I also reduced the potassium ferricyanide and potassium ferrocyanide to 1mM, and the Triton X-100 to 0.05%. I used 50mM Sodium citrate buffer pH 7.2 instead of phosphate buffer. Also, depending on the promoter that I was using, I had to incubate at 37C in the dark for up to 48h. But, at the end of the day, the results were beautiful.
Pollen Fertility/viability Assay Using FDA Staining
Author:  Xiyan Li, date: 05/20/2011, view: 30258, Q&A: 3
Pollen grains can be fertile or sterile by nature. This method stains pollen grains for an enzyme as the vital indicator of membrane integrity. Only fertile grains fluoresce under microscopic examination.
Very straightforward!
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