EdU staining

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

Invitrogen™ EdU (5-ethynyl-2'-deoxyuridine) ( Manual )

EdU ( 5-Ethynyl-2'-deoxyuridine)

Molecular weight: 252.22 g / mol

Solubility:

Solvent Maximum solubility Concentration

DMSO 25 mg / mL 100 mM

water 6.25 mg / mL 25 mM

aqueous buffer 6.25 mg / mL 25 mM

Solvent	Maximum solubility	Concentration
DMSO	25 mg / mL	100 mM
water	6.25 mg / mL	25 mM
aqueous buffer	6.25 mg / mL	25 mM

Materials NOT Provided

Please refer to the last page to make the PBS 10X stock and fixative solution.

Material	Concentration	Storage location
Phosphate buffer saline ( PBS )	1X	JK's bench
Fixative solution	4%	biosafety cabinet
Fluoromount-G	--	4°C ( d )

Procedure

Step 1: Labeling

Prepare incubation medium containing 10 µM Component A ( EdU );

The manufacturer's manual suggests to dilute the EdU from 10 mM to 10 µM for in vitro experiment. When EdU was dissolved in DMSO, this concentration of DMSO might facilitate the entry of organic molecules into tissue. However, 0.1% of DMSO will affect root growth. So, consider to make higher concentration of EdU stocks, change solvent, or reduce the working solution concentration.

Incubate the plants in the incubation medium in the same cultivation condition for 1 hour.

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

Prepare 200 µL fixative solution in eppendorf for each group of sample;
Cut the root tip ( around 1 cm ) and fully immerse in the fixative solution;

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

Incubate 30 minutes under room temperature;
Remove fixative solution and add 400 µL 1X PBS to wash the roots. Gently pipetting 5-10 times ( avoid touching the root tip ), then leave on the benchtop for 10 mins. Repeat this wash procedure three times ( 3 × 10 mins ).

Discard the fixative solution in the dark blue toxic carboy under the toxic cabinet;

(optional) You can keep the samples in 1X PBS solution up to 24 hours in 4°C protected from light after washing out the fixative solution.

Step 3: Detection

Prepare the cocktail

Place all the components on ice and protected from light. Prepare the cocktail as much as necessary only for that day's experiments, and use on the same day.

10X Component E is stored at −20°C in JK's white box.

the other components are stored at 4°C, in the kit box.

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.
The cocktail must be used 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

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

Remove wash solution and add 197 µL of Click-iT® reaction cocktail ( prepare as the table above ) for each sample;
Incubate for 30 minutes at room temperature. Must be protected from light;
Remove the reaction cocktail and wash once with 200 µL of Click-iT® reaction rince buffer ( Component F );
Wash 3 times with 1X PBS ( 3 × 10 mins );
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

Parameters	Theoretical value	Our machine
Excitation peak	495 nm	488 nm
Emission peak	519 nm	499 ~ 539 nm
Magnification		10X
Laser intensity ( 488 nm )		2.0%
Master Gain		600 V
Digital Gain		1.0
Pinhole		≈ 32 µm
Z-stack interval		2 µm
Scan speed		6
Scan direction		⟶

Step 5: Image processing

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

Open the confocal .czi file with the ImageJ, the "Bio-Formats Import Options" will automatically pop up;
Select the options as follow:

Options Choose

View stack with: Hyperstack

Color mode: Colorized

Autoscale ☑

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

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...

Save the images as TIFF format;

File → Save as → Tiff...

Select the region of interest (ROI) and measure.

Done !!!

Materials Provided by the kit

Component	Material name	C10351 ^*	Concentration	Location
A ^α	EdU working solution	525 µL	10 mM	4°C ( c )
B ^β	Alexa Fluor® azide 488	330 µL	1X	4°C ( c )
C ^γ	Click-iT® EdU reaction buffer	15 mL	10X	4°C ( c )
D ^δ	CuSO₄	1 vial	100 mM	4°C ( c )
E ^ε	Click-iT® EdU buffer additive	400 mg	10X	−20°C
F ^ζ	Click-iT® reaction rinse buffer	125 mL	1X	4°C ( c )

^* 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 B: Fluorescence probe. Protected from light.

^γ 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.

^δ Component D: Catalyze the reaction.

^ε 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.

^ζ Component F: Used to wash cells after the Click-iT® reaction cocktail incubation step. This helps to remove unreacted components and reduce background. Located at 4 °C ( c ), in front of the EdU kit boxes.

Materials NOT Provided by the kit

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

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 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

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

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