# **GUS staining**
X-GlcA, buy from [CyrusBioscience](http://cyrusbio.com.tw/webc/html/product/show.php?num=260&page=1&kw=X-Gluc)
> 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 !!!