# BES–H2O2–Ac
Hydrogen peroxide ( H2O2 ) detection probes.
> Manufacturer : Wako
> Molecular Formula : C28H11F7O8S
> M.W. = 640.44 g / mol
# Procedure
## Step 1: Prepare stock solution ( 10 mM )
Dissolve 1 mg powder in 156.2 µL DMSO to form a 10 mM stock solution. Store at room temperature, must be protected from light.
## Step 2: Stain
### Short time incubation
Dilute to 10 - 50 µM with growth medium. Incubate at room temperature for ≈ 20 mins, protected from light.
### Long time incubation
1. Make medium contains 100 nM BES-H2O2-Ac.
- Add 4 µL of 10 mM stock solution to 400 mL medium before the medium solidified ( ~ 50°C ), mix thoroughly, must be protected from light.
2. Grow the plant on this medium for ≈ 6 days, cut the root and observe using confocal microscope.
## Step 3: Confocal parameters
Set the confocal parameters as follow:
| Parameters | Theoretical value | Our machine |
|:-------------------------------------------------------- |:-----------------:|:--------------------: |
| Excitation peak | 485 nm | 488 nm |
| Emission peak | 515 nm | 505 ~ 541 nm |
| Magnification | | 5X |
| Laser intensity ( 488 nm ) | | 20.0% |
| Master Gain ( 488 nm ) | | 700 V |
| Master Gain ( T-PMT ) | | 240 V |
| Digital Gain | | 1.0 |
| Pinhole | | ≈ 35 µm |
| Z-stack interval | | 16 µm |
| Scan speed | | 6 |
| Scan direction | | ⟷ |
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## **Step 4: Image processing**
Open the CZI file with ImageJ, view stack with `Hyperstack`.
### 1. Generate BES and T-PMT layers
Repeat this process twice to generate both the BES and T-PMT layers:
`Image` ⟶ `Stacks` ⟶ `Z Project...` ⟶ Projection type: `Max intensity` ⟶ `File` ⟶ `Save As` ⟶ `Tiff...`
### 2. Generate stacks
Load in the previously generated BES and T-PMT TIFF files.
`Image` ⟶ `Stacks` ⟶ `Images to Stack`
### 3. Measure distance
Measure the distance between root tip and the BES signal distal boundary. The root tip can be easily recognized in the T-PMT layer.
Done !!!