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Plants Tissue-Clearing Reagent Suitable for Fluorescent Protein Observation: iTOMEI

For observing fluorescent protein clearly without complicated procedures

Improved TOMEI (iTOMEI) is a method that specializes TOMEI 1) for fluorescent protein observation by Prof. Sakamoto et al. This method enables simple transparency and allows for clear detection of fluorescent proteins.
TCI offers suitable reagents for iTOMEI.

Arabidopsis thaliana obtained using confocal microscope

Figure 1. Optical sections images of leaf of Arabidopsis thaliana obtained using confocal microscope.


Cleared Arabidopsis thaliana by iTOMEI

Figure 2. Cleared Arabidopsis thaliana by "iTOMEI"
(left) Non-treatment (right) iTOMEI treatment

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Advantages

  • Enables transparency by simple osmosis in just a few days (more than 2 days).
  • Keeps the fluorescence of a fluorescent protein such as GFP or tdTomato co-expressed as a reporter gene.
  • Suppresses autofluorescence.
  • Applicable to wide plant species including Oryza sativa, Arabidopsis thaliana, Marchantia polymorpha.

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Example of use

Preparation [Reagents]
  • 1% PFA/PBS (Preparation at time of use is recommended)
  • PBS
  • Decoloring solution (Tissue-Clearing Reagent iTOMEI-D [for Plants])
  • Mounting solution: Please use the suitable mounting solution for objective lens. Tissue-Clearing Reagent iTOMEI-M (RI 1.40) [for Plants] is optimized to silicone immersion objectives (reflactive index 1.40).
    (Iohexol solution is used in the example below.)
Fixing Fix the sample with 1% PFA/PBS for 1 hour at room temperature. *1
(Deaerate in fixative solution using vacuum pump or syringe when the sample is above ground part.)
Washing Remove PBS and add the decoloring solution, then let it gently shake for 24 hours with shading at room temperature. *1
Decoloring Remove the fixation solution and add PBS, then let it rest for 5 minutes at room temperature.
Repeat the same work twice. *1
Washing Remove the decoloring solution and add PBS, then let it rest for 5 minutes at room temperature.
Repeat the same work twice more.
Staining Remove PBS and add the staining solution, then let it rest with shading at room temperature. *2
Washing Remove the staining solution and add PBS, then let it rest for 5 minutes at room temperature.
Repeat the same work twice.
Clearing Remove PBS and add the mounting solution, then let it gently shake for 1 hour with shading at room temperature. *3
Mounting Mount the sample on a microscope slide with the mounting solution, seal by manicure and observe it.

*1 Operation time is determined by the sample size and plant type.

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

*3 If you want to moderate the change in osmotic pressure, it is necessary to perform a gradual replacement with a low-concentration mounting solution.

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Reagent enabling more rapid transparency (2 - 3 hours)

For observing only fluorescent staining dyes

T3530
Tissue-Clearing Reagent TOMEI [for Plants]

*T3530 is unavailable except in Japan, U.S. and Europe

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References

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