SCALEVIEW-A2
Dr. Atsushi Miyawaki et al. developed a water-based optical clearing reagent, Scale, which clears fixed biological samples withoutquenchingfluorescent protein. Using SCALEVIEW-A2, bodily tissues can be cleared of their opacity to provide a clearly defined image. The SCALEVIEW approach integrates seamlessly with multiphoton microscopes, providing the power to visualise 3-dimensional structures at unprecedented depths in morphologically intact tissue.
Optical characteristic : Refractive index ne(20℃) of SCALEVIEW-A2 is 1.377 - 1.381.
Protocol
Example: Rendering mouse brain tissue transparent for deep imaging according to reference1).
- Transcardially perfuse an anesthetised mouse with 4% paraformaldehyde (PFA)/PBS (pH 7.5～8.0).
- Remove the whole brain and subject it to post-fixation in 4% PFA/PBS at 4 °C for 10 hrs and cryo-protection in 20% sucrose/PBS at 4°C for 24 hrs.
- Embed the sample in OCT compound and freeze it with liquid nitrogen.
- Thaw and rinse the sample in PBS, and fix it again with 4% PFA/PBS for 20 min at room temperature.
- Clear the sample by incubation in SCALEVIEW-A2 at room temperature.
More than 30 ml of SCALEVIEW-A2 is required for an adult mouse brain.
A full week of incubation may be necessary for transparency.
During incubation, stir slowly using an orbital shaker.
Exchanging SCALEVIEW-A2 each day accelerates the transparency process.
The sample might be expanded 10-30% in one direction after incubation - Perform deep imaging of the transparent brain using an appropriate dipping objective lens.Use SCALEVIEW-A2 as the immersion medium.
- The transparent brain can be stored for a long time in SCALEVIEW-A2 at 4°C.
1.Image of a mouse brain after SCALEVIEW-A2 process
Figure 1
Amouse brain after SCALEVIEW-A2 process
Left: a mouse brain before SCALEVIEW-A2 process
Right: a mouse brain after SCALEVIEW-A2 process.
2. Imaging data using SCALEVIEW-A2
Figure 2
Imaging of Thy1-YFP(H Line) mouse brain after SCALEVIEW-A2 process using Multiphoton microscope and Multiphoton dedicated objective :OLYMPUS, model:XLPLN10XSVMP.Bars, 50μm.
Image
Imaging data using SCALEVIEW-A2
Figure 3
Deep imaging of Thy1-YFP(H Line) mouse brain after SCALEVIEW-A2 process using Multiphoton microscope and Multiphoton dedicated objective :OLYMPUS,
model:XLPLN25XSVMP(NA1.0 WD4mm).
Figure 4
Image of mouse pancreas(labeling process the blood vessel using Lectin Texas Red) after SCALEVIEW-A2 process using Confocal microscope and objective :OLYMPUS, UPLSAPO30XS..
Reference
1) Hama,H.et al. : Nature Neuroscience 14, 1481(2011).
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