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.
  1. 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 ⟷

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 !!!