EdU staining

Invitrogen™ Click-iT™ EdU Alexa Fluor™ 488 HCS Assay ( Manual )

5-Ethynyl-2'-deoxyuridine
252.22 g/mol
Maximum solubility: 25 mM in water and up to 100 mM in DMSO.

• "ddH₂O" means deionized ultrapure water;
• "complete medium" means the medium used to cultivate the samples, for example, half-MS medium in this case.

Procedure

Step 1: Labeling

1. Prepare incubation medium containing 10 µM Component A;

• For example, if we have five groups of samples, add 100 µL 10 mM Component A into 100 mL half-MS medium for the experiment. Then, dispense the diluted 10 µM Component A to new falcon tubes, each tube takes around 20 mL for each sample ( make sure the root tips can be fully submerged in the solution).

2. Incubate the plants in the incubation medium in the same cultivation condition for 1 ~ 2 hours.

• Directly immersed the roots into the solution, DO NOT cut the shoot part, the plant should still alive. The root should be fully submerged into the solution. Do not over-pushing the root to avoid roots injury.

Step 2: Fixation

1. Prepare 200 µL fixative solution for each sample ( per eppendorf );

2. Cut the root tip ( around 5 mm ) and fully immerse in the fixative solution;

• Better to do this in biosafety cabinet. If too many roots, better to cut the root on a rigid agar plate (~ 1%), and keep the surface wet with 1X PBS to prevent the root being dried.

3. Incubate 30 minutes under room temperature;

4. Remove fixative solution and add 400 µL 1X PBS to wash the roots. Gently pipetting 5-10 times ( avoiding contact with the root tip ), then leave on the benchtop for 10 mins. Repeat this wash procedure three times ( 3 × 10 mins ).

• You can keep the samples in 1X PBS solution for at least 24 hours in 4°C protected from light after removing the fixative solution.

Step 3: Detection

Prepare the cocktail

1. Dilute the Component C and Component E from 10X to 1X using ddH₂O. Calculate the total usage amount according to the following tables.

• 10X Component C is stored at 4°C in the kit box.
• 10X Component E is stored at -20°C in JK's white box.
• Prepare the Component C and E as much as necessary only for that day's experiments, and use on the same day.

2. Add the Click-iT® reaction cocktail ingredients as follow.

• Add the incredients in the order listed in the table; otherwise, the reaction will not proceed optimally.
• Use the cocktail immediately after preparation. The Click-iT® reaction buffer additive is susceptable to oxidation and is the limiting factor to the Click-iT® reaction cocktail's effectiveness over time.

Component	Material name	Addition per sample	Note
ddH2O	deionized ultrapure water	171 µL
C	10X Click-iT® EdU reaction buffer	17 µL
D	CuSO4	8 µL
B	Alexa Fluor® azide	0.5 µL	keep in dark
E	10X Click-iT® EdU buffer additive	2 µL	add before use
Total:		198.5 µL	take 197 µL

Incubation

3. Remove wash solution and add 197 µL of Click-iT® reaction cocktail ( prepare as the table above ) for each sample;
4. Incubate for 30 minutes at room temperature. Must be protected from light;
5. Remove the reaction cocktail and wash once with 200 µL of Click-iT® reaction rince buffer ( Component F );
6. Wash 3 times with 1X PBS ( 3 × 10 mins );
7. Mount on slide with Fluoromount-G anti-fade solution^†. Proceed to confocal imaging and analysis.

^† Using ddH₂O is also fine if the laser intensity is low and the laser exposure time ( image capturing time ) is short.

Step 4: Confocal parameters

Step 5: Image processing

1. Use ImageJ with bioformats_package.jar plugin to proceed the confocal images;

• The plugin ( bioformats_package.jar ) can be downloaded from https://www.openmicroscopy.org/bio-formats/downloads/

• The plugin should be placed in the "/ImageJ/plugins/jars" directory

2. Open the confocal .czi file with the ImageJ, the "Bio-Formats Import Options" will automatically pop up;

3. Select the options as follow:

Options	Choose
View stack with:	Hyperstack
Color mode:	Colorized
Autoscale	☑

4. Stack the image layers and perform max intensity projection along the Z-axis;

• Image → Stacks → Z project... → Projection type: Max Intensity

Optional: Shows scale bar in the image.
Analyze ⟶ Tools ⟶ Scale Bar...

5. Save the images as TIFF format;

• File → Save as → Tiff...

6. Select the region of interest (ROI) and measure.

Step 6: Data analysis

Finished !!!

Materials Provided by the kit

^* C10351: Catalogue number. All the raw materials in this kit should be stored at 2 ~ 6°C, dessiccated, protect from light, and DO NOT FREEZE.

^α Component A: This is the EdU chemical stock solution. Dilute to 10 µM in complete medium on the day of the experiment, and use immediately. The 10 mM stock solution is stored at -20°C ( the EdU powder also put in -20°C, in JK's white box ), and the 10 mM aliquots are stored at 4°C (in the EdU kit box, put together with the other components).

^β Component C: Dilute from 10X to 1X using ddH₂O, i.e., 15 mL 10X Component C + 135 mL ddH₂O.
The 1X solution could be stored at 2 ~ 6°C for 6 months.

^γ Component E: Add 2 mL ddH₂O to the vial of the Component E, mix until fully dissolve the powder to 10X solution. The 10X solution could be stored at ≤ −20°C for up to 1 year. If the solution develops a brown color, it has degraded and should be discarded.

Materials NOT Provided

^δ 1X PBS ( pH 7.4 )

• Make 10X PBS stock solution first as follow ( 10X stock located at 4°C bottom right ). When in use, dilute to 1X PBS.

10X PBS contents	M.W. (g/mol)	Addition
NaCl	58.44	80.1 g
KCl	74.55	2.0 g
Na2HPO4	141.96	14.4 g
KH2PO4	136.09	2.7 g
ddH2O		1 L

• 10X PBS ⟶ Adjust to pH 7.4 ⟶ Autoclave ⟶ Dilute to 1X ( 100 mL 10X PBS + 900 mL ddH₂O )

^ε Fixative solution

Chemical	Addition	Final concentration	Storage location
Formaldehyde (38%)	105 µL	4%	Toxic cabinet D
Triton X-100 (100%)	1 µL	0.1%	IV
1X PBS	894 µL
Total:	1000 µL