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Tissue Optical Clearing Reagent
Take a Revolutionary Approach to Deep Image

SCALEVIEW-S

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.

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

1. Link

2. Benefits of SCALEVIEW-S

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

3. SCALEVIEW-S Applications

  Fixation Clearing Imaging
Perfusion fixation + post fixation
4% PFA/PBS(-) [pH 7.6 - 7.8]
Step 1.
SCALEVIEW®-S0
Step 2.
SCALEVIEW®-S1
Step 3.
SCALEVIEW®-S2
Step 4.
SCALEVIEW®-S3
Step 5.
deScale Solution
Step 6.
SCALEVIEW®-S4
Step 7.
SCALEVIEW®-SMt
Mounting
SCALEVIEW®-SMt
Processing temperature 4 °C 37 °C 37 °C 37 °C 37 °C 4°C 37 °C 37 °C RT
Processing time 3 days 30 min 30 min 30 min 30 min 3 hrs x 2 12 - 24 hrs 1 hr  

 

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

 

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

4. SCALEVIEW-S Applications: IHC (AbScale)

  Fixation Preprocessing
Perfusion fixation + post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®-S0
Step 2.
SCALEVIEW®-S1
Step 3.
SCALEVIEW®-S2
Step 4.
SCALEVIEW®-S3
Step 5.
deScale Solution
Processing temperature 4 °C 37 °C 37 °C 37 °C 37 °C 4°C
Processing time 3 days 4 hrs 4 hrs 12 hrs 4 hrs 6 hrs
  IHC Clearing Imaging
Step 1. Blocking
1% Blocking
Reagent (Roche)/PBS(-)
Step 2. Staining
Antibody stains
Step 3. Wash
AbScale Solution
Step 4. Re-fixation
4% PFA/PBS(-)
[pH7.6 - 7.8]
Step 5. Wash
deScale Solution
SCALEVIEW®-S4 Mounting:
SCALEVIEW®-S4
Processing temperature RT 37 °C RT 4°C 4°C 37 °C RT
Processing time 2 hrs > 1day 2 hrs x 1,
1 hr x 1
1 hr 6 hrs 6 - 8 hrs  

*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)

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)

5. SCALEVIEW-S Applications: Fluorescent labels (ChemScale)

  Fixation Preprocessing
Perfusion fixation + post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®-S0
Step 2.
SCALEVIEW®-A2
Step 3.
8M Urea Solution
Step 4.
SCALEVIEW®-A2
Step 5.
deScale Solution
Processing temperature 4 °C 37 °C 37 °C 37 °C 37 °C 4°C
Processing time 3 days 4 hrs 4 hrs 12 hrs 4 hrs 6 hrs
  Fluorescent labels Clearing Imaging
Step 1. FL labels
ex:DAPI(500nM), PI(1μg/mL)/SCALEVIEW®-A2
Step 2. Wash
SCALEVIEW®-A2
Step 3. Wash
deScale Solution
SCALEVIEW®-S4 Mounting:
SCALEVIEW®-S4
Processing temperature 37 °C 37 °C 4 °C 37 °C RT
Processing time 6 -8 hrs 2 hrs x 1,
1 hr x 1
3 hrs 6 - 8 hrs  

 

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 ) 

6. SCALEVIEW-S Applications: Neurosphere

  Fixation Preprocessing
Perfusion fixation + post fixation
4% PFA/PBS(-)
[pH 7.6 - 7.8]
Step 1.
SCALEVIEW®-S0
Step 2.
SCALEVIEW®-A2
Step 3.
8M Urea Solution
Step 4.
SCALEVIEW®-A2
Step 5.
deScale Solution
Processing temperature RT 37 °C 37 °C 37 °C 37 °C 4°C
Processing time 1 hr 4 hrs 4 hrs 12 hrs 4 hrs 6 hrs
  IHC Clearing Imaging
Step 1. Blocking
1% Blocking
Reagent (Roche)/PBS(-)
Step 2. Staining
Antibody stains
Step 3. Wash
deScale Solution
Step 4. Re-fixation
4% PFA/PBS(-)
[pH7.6 - 7.8]
Step 5. Wash
deScale Solution
SCALEVIEW®-S4 Mounting:
SCALEVIEW®-S4
Processing temperature RT 37 °C RT RT 4°C 37 °C RT
Processing time 2 hrs > 1day 2 hrs x 1,
1 hr x 1
1 hr 3 hrs 4 hrs  

 

Figure5. 3D visualization of Neurosphere

Microscope (CLSM) Olympus FV1000
Objective lens

UMPLFLN10XW (NA 0.3)

7. 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 Reports8, 11135 (2018).

8. Product list

Product Name Grade Pkg. Size Wako Cat. No. Storage Condition
SCALEVIEW-S0 Tissue Optical Clearing Reagent 250 mL

196-18521

Keep at 2-10°C
SCALEVIEW-S1 Tissue Optical Clearing Reagent 250 mL

193-18531

Keep at 2-10°C
SCALEVIEW-S2 Tissue Optical Clearing Reagent 250 mL

190-18541

Keep at 2-10°C
SCALEVIEW-S3 Tissue Optical Clearing Reagent 250 mL

197-18551

Keep at 2-10°C
SCALEVIEW-S4 Tissue Optical Clearing Reagent 250 mL

194-18561

Keep at 2-10°C
SCALEVIEW-SMt Tissue Optical Clearing Reagent 250 mL

191-18571

Keep at 2-10°C
deScale Solution Tissue Optical Clearing Reagent 500 mL 041-34425 Keep at 2-10°C
SCALEVIEW-S Trial Kit Tissue Optical Clearing Reagent 1 kit 299-79901 Keep at 2-10°C
SCALEVIEW–A2 Tissue Optical Clearing Reagent 500 mL

193-18455

Keep at RT
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