Tissue-Clearing Reagent TOMEI [for Plants]

The transparency technique “TOMEI-I” is adequate for observation with only fluorescent dyes such as DAPI staining, Calcofluor White staining, etc.

【Reagents】
・Fixative solution (Acetic acid : Ethanol = 1 : 3) *Preparation at time of use is recommended.
・PBS
・70% Ethanol (diluted with PBS)
・30% Ethanol (diluted with PBS)
・Staining solution
・Transparency reagent TOMEI (TCI product code T3530)
*For root specimens, 20% TOMEI diluted with PBS is necessary.

【Procedure】
The transparency method of the final process differs depending on the above ground part and the underground part (root).

< Fixing >
1) Fix the sample with adequate amount of fixative solution at room temperature.
(Fixation time is determined by the sample size and plant type.)
(In case of a seedling of Arabidopsis thaliana, it usually takes for about 1 to 2 hours.)
2) Remove fixation solution and add 70% ethanol, then let it rest for 5min at room temperature.
3) Remove 70% ethanol and add 30% ethanol, then let it rest for 5min at room temperature.
4) Remove 30% ethanol PBS and add PBS, then let it rest for 5min at room temperature.

< Staining >
5) Remove PBS and add the staining solution, then let it rest with shading at room temperature. *1
(In case of double staining, re-stain after washing.)

< Washing >
6) Remove the staining solution and add PBS, then let it rest for 10min at room temperature.
7) Remove PBS and add PBS, then let it rest for 10min at room temperature.
8) Remove PBS and add PBS, then let it rest for 10min at room temperature.

< Clearing >
* For above ground part
9) Remove PBS and add TOMEI, then let it rest with shading at room temperature for 20min.
(The time to treat should be considered depending on sample.)
10) Mount the sample on a microscope slide with TOMEI and observe it.
*For underground part (root)
9) Remove PBS and add 20% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
10) Mount the sample on a microscope slide with 20% TOMEI in PBS and observe it.

*1 : The treatment time of DAPI staining is for 30min with 5 μg/mL and the treatment time of Calcofluor White staining is for 10min with 1 g/L of Calcofluor White M3R and 0.5 g/L of Evans Blue, but their adjustments are needed for the purpose.

References

• Three-Dimensional Imaging of Plant Organs Using a Simple and Rapid Transparency Technique
  • J. Hasegawa, Y. Sakamoto, S. Nakagami, M. Aida, S. Sawa, S. Matsunaga, Plant Cell Physiol. 2016, 57, 462.

The transparency technique “TOMEI-II” is adequate for observation fluorescent protein such as GFP or YFP co-expressed as a reporter gene.

【Reagents】
・Fixative solution (4% paraformaldehyde in PBS)
 *Preparation at time of use is recommended.
・PBS
・Staining solution
・Transparency reagent TOMEI (TCI product code T3530)
・10%, 30%, 50% , 70% TOMEI (diluted with PBS)
 *For the root specimens, 20% TOMEI diluted with PBS is necessary.

【Procedure】
The transparency method of the final process differs depending on the above ground part and the underground part (root).

< Fixing >
1) Fix the sample with adequate amount of fixative solution at room temperature.
 (Fixation time is determined by the sample size and plant type.)
 (Deaerate in fixative solution using vacuum pump or syringe when the sample is above ground part.)
2) Remove fixation solution and add PBS, then let it rest for 5min at room temperature.
3) Remove PBS and add PBS again, then let it rest for 10min at room temperature.
4) Remove PBS and add PBS again, then let it rest for 10min at room temperature.

< Staining >
5) Remove PBS and add the staining solution, then let it rest with shading at room temperature.*1
 (In case of double staining, re-stain after washing.)

< Washing >
6) Remove the staining solution and add PBS, then let it rest for 10min at room temperature.
7) Remove PBS and add PBS, then let it rest for 10min at room temperature.
8) Remove PBS and add PBS, then let it rest for 10min at room temperature

< Clearing >
*For above ground part
9) Remove PBS and add 10% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
10) Remove 10% PBS and add 30% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
11) Remove 30% TOMEI in PBS and add 50% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
12) Remove 50% TOMEI in PBS and add 70% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
13) Remove 70% TOMEI in PBS and add 100% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
14) Remove 100% TOMEI in PBS and add 100% TOMEI, then let it rest with shading at room temperature for 1hour.
 (The time to treat should be considered depending on sample.)
15) Mount the sample on a microscope slide with TOMEI and observe it. *2
*For underground part (root)
9) Remove PBS and add 20% TOMEI in PBS, then let it rest with shading at room temperature for 10min.
10) Mount the sample on a microscope slide with 20% TOMEI in PBS and observe it.

*1 : The treatment time of DAPI staining is for 30min with 5 µg/mL and the treatment time of Calcofluor White staining is for 10min with 1 g/L of Calcofluor White M3R and 0.5 g/L of Evans Blue, but their adjustments are needed for the purpose. *2 : When the fluorescence of fluorescent protein reduces, after the treatment of 50% TOMEI in PBS, it may be possible to be solved by being mounted with 60% TOMEI in PBS after the treatment of 60% TOMEI in PBS for 1hour.