# **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 |
> ||||
# 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 ) |
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# Procedure
## **Step 1: Labeling**
1. Prepare incubation medium containing 10 µM Component A ( EdU );
- Component A ( 100 µM stock ) is stored at 4°C ( e-bottom-left ) JK's green box ( top-left corner side ).
- Generally we make 40 mL incubation medium in 50 mL falcon tube for each treatment groups. For example, if we have 6 treatment groups, add 25 µL Component A stock ( 100 µM ) into 250 mL medium and mix thoroughly. Then, dispense 40 mL to each tube.
> 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. Currently, this protocol uses higher concentration of EdU stock ( 100 µM ), and the staining result is statisfiying.
2. 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. Fully submerged the root into the solution. Do not over-pushing, avoid roots injury.
## **Step 2: Fixation**
1. Prepare 400 µL fixative solution in eppendorf for each group of sample;
2. 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.
3. Incubate 24 hours at room temperature;
- Incubation time can be varying from 30 minutes to 24 hours, depends on the sample condition. The longer the incubation time, the weaker the background autofluorescence and the EdU signal. When dealing with thick tissues, short incubation time may results incomplete penetration of the fixative solution, background autofluorescence, and too-strong EdU signal (false positive??). We incubate 24 hours to achieve clean background and optimal EdU signal intensity.
4. 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 around 5 times ( 5 × 10 mins ) until no bubbles appear.
- Discard the fixative solution in the dark blue toxic carboy under the toxic cabinet;
5. (optional) You can keep the samples in 1X PBS solution up to 48 hours in 4°C protected from light after washing out the fixative solution.
## **Step 3: Detection**
### **Prepare the cocktail**
1. 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.
> - Component B, C, and D are stored at 4°C, in the kit box.
> - Component E ( 10X stock ) is stored at −20°C in JK's white box.
> - Component F is stored at 4°C, placed in front of the kit box.
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.
- 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 195 µL |
### **Incubation**
3. Remove wash solution and add 195 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 400 µL of Click-iT® reaction rince buffer ( Component F ) ( 1 × 15 mins );
6. Wash 5 times with 1X PBS ( 5 × 15 mins );
- You can keep the samples in 1X PBS solution or reaction rince buffer ( Component F ) up to 24 hours in 4°C and protected from light after washing out the cocktail. If kept more than 24 hours, background fluorescence could be quite obvious. Before mount on slide, wash the sample again with 1X PBS for at least twice ( 2 × 15 mins ) to minimize background autofluorescence.
7. Proceed to confocal imaging and analysis.
## **Step 4: Confocal parameters**
Mount on slide with Fluoromount-G anti-fade† solution. Using ddH2O is also fine if the laser intensity is low and the laser exposure time ( image capturing time ) is short.
† Located at 4°C (d).
Set the confocal parameters as follow:
| Parameters | Theoretical value | Our machine |
|:-------------------------------------------------------- |:-----------------:|:--------------------: |
| Excitation peak | 495 nm | 488 nm |
| Emission peak | 519 nm | 499 ~ 539 nm |
| Magnification | | 10X |
| Laser intensity ( 488 nm ) | | 16.0% |
| Master Gain | | 700 V |
| Digital Gain | | 1.0 |
| Pinhole | | ≈ 32 µm |
| Z-stack interval | | 4 µm |
| Scan speed | | 6 |
| Scan direction | | ⟷ |
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## **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.
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 ) |
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* **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 stock should be dispense into several aliquots ( 400 µL each eppendorf for example ) to avoid too frequent thawing and freezing, to prolong the storage lifespan. 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 |
> ||||
## 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 | | |