Dr. Takeshi Imai. et al. developed a water-based optical clearing reagent, SeeDB (See Deep Brain), which clears fixed brain samples in a few days without quenching many types of fluorescent dyes, including fluorescent proteins and lipophilic neuronal tracers. SeeDB is a saturated solution of fructose in water with thioglycerol. This method facilitates comprehensive and quantitative analyses for understanding neuronal circuitry, both in the adult and developing mouse brain.
Image data courtesy of：Takeshi Imai,Meng-Tsen Ke, Laboratory for Sensory Circuit Formation RIKEN Center for Developmental Biology
- Fix the sample in 4% PFA at 4 °C with gentle shaking overnight.
- Wash the sample in PBS three times (10 min each).
- Incubate the sample in ~20 mL of SeeDB:20w/v% Fructose Solution in 50 mL conical tube, and then place the conical tube on a tube rotator (~4 rpm) or a seesaw shaker (~17 rpm) for 4-8 h, respectively.
A small piece of sample (e.g., slices) requires less time for optical clearing. Incubation should be performed at 25-37 °C.
- Incubate the sample in ~20 mL SeeDB:40w/v% Fructose Solution for 4-8 h as above.
- Incubate the sample in ~20 mL SeeDB:60w/v% Fructose Solution for 4-8 h.
(Samples may no longer sink in 60% or higher concentrations of fructose.)
- Incubate the sample in ~20 mL SeeDB:80w/v% Fructose Solution for 12 h.
- Incubate the sample in ~20 mL SeeDB:100w/v% Fructose Solution for 12 h.
- Incubate the sample in ~20 mL SeeDB for 24 h. The incubation time can be extended up to 48 h.
- The transparency can be evaluated by eye at this stage . If the sample is successfully cleared, the adult brain sample should look amber under a light source.
- Observe the SeeDB-treated samples under a fluorescence microscope.
Samples should be mounted in SeeDB solution.
Image of a mouse brain after SeeDB process
Figure.1 Mouse embryo and mouse brain before and after treatment with SeeDB
Imaging data using SeeDB
Figure2. Imaging of Thy1-YFP(H Line) mouse brain after SeeDB process using Multiphoton microscope and Multiphoton dedicated objective :OLYMPUS, model:XLPLN10XSVMP.
Figure 3. Imaging of Thy1-YFP(H Line) mouse brain after SeeDB process using Confocal microscope and objective :OLYMPUS, UPLSAPO 10X2.
Figure 4. Imaging of Thy1-YFP(H Line) mouse brain after SeeDB process using Multiphoton microscope and Multiphoton dedicated objective :OLYMPUS, model: XLSLPLN25XGMP. Bars, 50μm.
Figure.5 Imaging of DiI-labeled mitral cells in the olfactory bulb after SeeDB process using Confocal microscope and objective :OLYMPUS, UPLSAPO 20X. Bars, 100 µm.
- Ke, M. T., Fujimoto, S. and Imai, T. : Nat Neurosci, 16 (8), 1154(2013).
- Ke, M. T., Fujimoto, S. and Imai, T.: Bio-protocol, 4(3), e1042(2014).
- Ke, M. T., and Imai, T. : Curr Protoc Neurosci, 66, 2.22.1-2.22.19(2014).
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