Take a Revolutionary Approach to Deep Image

SCALEVIEW-S

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Data provided by Dr. Hiroshi Hama, Tetsushi Hoshida and Dr. Atsushi Miyawaki, Laboratory for Cell Function Dynamics, Brain Science Institute, RIKEN Biotechnological Optics Research Team, Center for Advanced Photonics, RIKEN Cooperation with Olympus

The original recipe reported by the Miyawaki team in 2011 termed Scale was an aqueous solution based on urea that limited because the transparency process itself can damage the structures under study.
The research team spent 5 years improving the effectiveness of the original recipe to overcome this critical challenge, and the result is ScaleS, (we called SCALEVIEW-S) a new technique with many practical applications. SCALEVIEW-S creates transparent brains with minimal tissue damage, that can handle both florescent and immunohistochemical labeling techniques, and is even effective in older animals.
The new technique creates transparent brain samples that can be stored in SCALEVIEW-S solution for more than a year without damage. Internal structures maintain their original shape and brains are firm enough to permit the micron-thick slicing necessary for more detailed analyses.

Features

  • Easy-to-use
  • No special equipment required
  • Less damage to sample
  • Compatible with IF, FP and other fluorescent labels

[Use it for...]
Mouse brain, human post-mortem brain, bone*, organoid/spheroid
*Decalcification is required for bone.

Necessaries(when using SCALEVIEW-S Trial Kit)

Reagents Instrument
  1. SCALEVIEW-S Trial Kit
  2. deScale Solution (Code No. 041-34425)
  3. Paraformaldehyde (Code No. 160-16061)
  4. 1×PBS (Code No. 164-25511)
  5. Agarose
  1. 5 mL/15 mL/30 mL/50 mL conical centrifuge tube
  2. Culture dish (100 mm, 60 mm and 35 mm diameter) (for each sample)
  3. Seesaw shaker
  4. Fluorescence microscope and Recommend Objective lenses for SCALEVIEW-S treated samples

Protocols

Protocol for clearing of brain slices (thickness: 1-2mm)

It is recommended to prepare slices after fixation process.

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Figure 1. Transmittance images of mouse brain before and after clearing with SCALEVIEW-S Solutions.

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Figure 2. Two-photon microscope imaging of YFP-H line: Mouse whole brain

Mouse Thy1-YFP-H line, 20W, ♂
Size Whole
Microscope Olympus FVMPE-RS
Objective lens XLPLN10XSVMP (NA 0.6)
Laser 960 nm (for YFP)
Image size 512 x 512, 170 tiles, Z=8000 μm, Z Step16 μm

Protocol for 3-D IHC (AbScale)

It is recommended to prepare slices after fixation.

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**AbScale Solution:0.33 M Urea and 0.1 % (wt/vol) Triton X-100 in PBS(-) Solution

Iba1 (RF635: Green)  Amyloid-β (Alexa Fluor 488: Red)  Tomato lectin (Texas Red: Blue)

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Figure 3. 3D visualization of Aβ plaques (red), microglias (green) and blood vessels (blue) from a 17-month-old AD model mouse.

Microscope (CLSM) Olympus FV1200
Objective lens XLPLN10XSVMP (NA 0.60)

Protocol for 3-D Chemical Staining (ChemScale)

It is recommended to prepare slices after fixation.

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Figure 4. Confocal laser scanning microscope imaging of fluorescent labeled (PI) YFP-H line mouse brain slice (2 mm thick).

Mouse Thy1-YFP-H line, 42W, ♂ Microscope (CLSM) Olympus FV3000 (Inverted)
Size Coronal Slice (2 mm) Objective lens UPLSAPO10x2 (NA 0.40)
Laser 488 nm (for YFP), 561 nm (for PI )

Protocol for AbScale of neurosphere

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*AbScale Solution:0.33 M Urea and 0.1 % (wt/vol) Triton X-100 in PBS(-) Solution
** After clearing, embed and immobilize with 1.5% (wt/vol) agarose.

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Figure5. 3D visualization of Neurosphere

Microscope (CLSM) Olympus FV1000
Objective lens UMPLFLN10XW (NA 0.3)

References

  1. Hama,H.et al. : Nature Neuroscience 14, 1481(2011).
  2. Hama,H.et al. : Nature Neuroscience 18, 1518(2015).
  3. Hama H, et al. : Protocol Exchange (2016), doi:10.1038/protex.2016.019
  4. Molly E, Boutin, et al :Scientific Reports, 8, 11135 (2018).

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