GUS staining

X-GlcA, buy from CyrusBioscience

IUPAC name: 5-Bromo-4-chloro-1H-indol-3-yl β-D-glucopyranosiduronic acid
Other name: 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide
Molucular formula: C14H12BrClNO7Na
Molecular weight: 444.6 g/mol

Procedure

Step 1: Prepare sodium phosphate buffer

50 mM, pH 7.0
Calculator

Chemical M.W. (g/mol) Addition Storage location
NaH2PO4 119.98 249.0 mg Ⅲ
Na2HPO4 141.96 414.0 mg Ⅲ
ddH2O 100 mL

Step 2: Prepare X-GlcA stock solution (100 mM)

Dissolve 444.6 mg of X-GlcA in 10 mL N,N-Dimethylformamide (DMF) or DMSO.
I keep the stock solution in −20°C JK's white box. Must be protected from light.

Chemical M.W. (g/mol) Addition Storage location
X-GlcA 444.60 444.6 mg −20°C JK's white box
DMF 10 mL Toxic (D)

Step 3: Prepare GUS stain working solution

The working solution must be fresh-made and use immediately after prepared.

1 mM X-Gluc
50 mM sodium phosphate buffer (Na-P) (pH 7.0)
7% methanol
0.1% TritonX-100

Chemical Concentration Addition Storage location
X-GlcA stock solution† 100 mM 1 mL −20°C
Methanol 100% 7 mL Toxic (D)
TritonX-100 100% 1 drop II
Sodium phosphate buffer ( pH 7.0 ) 50 mM 92 mL

† The stock solution is stored in the door compartment of the −20°C freezer in a black 10 mL tube.

Step 4: GUS Stain

  1. Fully immersed the rice seedling in the GUS stain solution;

  2. Vacumm infiltration at room temperature for 30 mins to allow the solution penetrate completely into the tissue;

  3. Incubate at 37°C in dark for 30 mins;

  4. (optional) Rinse with 75% or 95% ethanol until the chlorophyll was bleached;

  5. Rinse with water to fully wash out the stain solution;

  6. Observe under a dissection microscope.


Done !!!