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  5. SCALEVIEW
Take a Revolutionary Approach to Deep Image

SCALEVIEW

The SCALEVIEW series are reagents designed for use in the Scale method. Developed by Dr. Atsushi Miyawaki and his colleagues at RIKEN, the Scale method is an optical clearing technique that employs urea-based reagents. This method is not only useful for clearing biological samples containing fluorescent proteins but also significantly enhances the efficiency of labeling thick samples with antibodies and fluorescent dyes. It is particularly useful as an analytical tool for studying brain tissues, spheroids, and organoids.

What is Scale?

00593_img01.png

PI-stained brain hemisphere slice from a YFP-H mouse
Data by courtesy of
Drs. Hama, Hoshida, and Miyawaki of Laboratory for Cell Function Dynamics, RIKEN Center for Brain Science, and Biotechnological Optics Research Team, RIKEN Center for Advanced Photonics (With support from Evident)

Overview

Organic solvents have been widely used in conventional clearing techniques; however, fluorescent proteins that depend on water molecules for fluorescence emission tend to fade significantly. To address this issue, the Scale method (a collective term for the ScaleS and ScaleA2 methods) was developed1-2). This technique uses water-based reagents composed of urea, surfactants, and either sorbitol (in the ScaleS method) or glycerol (in the ScaleA2 method) to suppress light scattering, enabling tissue clearing without fading of fluorescent proteins. Moreover, the low surfactant content required for lipid removal helps maintain ultrafine structures after clearing. With its exceptional balance of transparency and structural preservation, the Scale method is highly recommended for those new to tissue clearing.

Features

  • Simple operation (just immerse samples sequentially in the solutions)
  • Clears 2 mm brain tissue blocks in about one day
  • Compatible with fluorescent dye staining (ChemScale) and Immunostaining (AbScale or AbScale-G)

Samples suitable for clearing

Mouse brain, Postmortem human brain, Bone (requires decalcification), Organoids/Spheroids, and more.

Selection of SCALEVIEW Series

Applications Fluorescent proteins Fluorescent dyes Antibody (Immunostaining)
Methods ScaleS ChemScale AbScale AbScale-G
Required Reagents SCALEVIEW-S Trial Kit ✔
SCALEVIEW-A2 ✔ ✔ ✔
SCALEVIEW-S0 ✔ ✔ ✔
SCALEVIEW-S4 ✔ ✔ ✔
descale solution ✔ ✔ ✔ ✔
AbScale-G Solution Kit ✔
Time required for clearing Approx. 1 day 4 to 7 days 4 to 7 days 4 to 7 days
Note •Compatible with co-staining of fluorescent proteins •Compatible with co-staining of fluorescent proteins •Compatible with co-staining of fluorescent proteins
•Applicable to antibodies and fluorescent dyes
(e.g., Cy3) due to enhanced permeability compared to AbScale.

Description of Each Reagent

SCALEVIEW-S Trial Kit

A kit containing six solutions: SCALEVIEW-S0, SCALEVIEW-S1, SCALEVIEW-S2, SCALEVIEW-S3, SCALEVIEW-S4, and SCALEVIEW-SMt. Sequential immersion of tissue samples in these solutions allows for simple and rapid tissue clearing and refractive index adjustment.

SCALEVIEW-A2

A clearing solution based on the Scale method reported in 2011. With a relatively low refractive index (RI = 1.377- 1.381 at 20°C), it allows easy refractive index matching even without expensive microscopy equipment, making it ideal for those new to tissue clearing.

descale solution

A reagent used in the washing process of the ScaleS method.

Simplified Spheroid Clearing (Optional)

A simplified clearing protocol for spheroids using only SCALEVIEW-S4 has been reported3).
Details of the protocol and experimental data for simplified clearing are available on the SCALEVIEW-S4 product details page.

Other Required Equipment, Instruments, and Reagents

Equipment and Instruments

  • Fluorescence microscope

    Ensure that the objective lenses are compatible with the refractive indices (SCALEVIEW-A2: RI=1.38, SCALEVIEW-S4: RI=1.47, SCALEVIEW-SMt: RI=1.49). Consulting the microscope manufacturer before data acquisition is recommended.

  • Conical tubes (choose from among 5 mL, 15 mL, 30 mL, or 50 mL tubes based on the sample)
  • Plastic petri dishes (choose from among 35 mm, 60 mm, or 100 mm diameter dishes based on the sample)
  • See-Saw Rocking Shakers

Reagents

  • Paraformaldehyde (Product Number: 160-16061)
  • 1 x PBS (Product Number: 164-25511)
  • Agarose

Data

Brain Tissue

ScaleS
Clearing Mouse Brain Sections and Hemispheres
00593_img12.png

Protocol

Perfusion fixation + Post-fixation Clearing Observation
4% PFA/PBS(-) PBS(-) SCALEVIEW-S0 SCALEVIEW-S1 SCALEVIEW-S2 SCALEVIEW-S3 descale Solution SCALEVIEW-SMt SCALEVIEW-SMt
4°C
3 days		 RT
Several times		 37°C
30 minutes		 37°C
30 minutes		 37°C
30 minutes		 37°C
30 minutes		 4°C
3 hour x 2 times		 37°C
1 hour		 RT

Volume of Each Solution Used (Approximate volumes for 1-2 mm thick mouse brain hemisphere sections*)

[Tissue Clearing] SCALEVIEW-S0/S1/S2/S3: 5 mL each / descale Solution 5 mL
[Observation] SCALEVIEW-SMt: 5 mL

* The volume of reagents and processing time vary with sample size.

　

ScaleS
Clearing the Whole Brain of YFP-H Mice
00593_img13.png

Species: Mouse (Thy1-YFP-H line, 20W, Male)
Tissue: Whole brain
Microscope: Multiphoton microscope (Evident, FVMPE-RS)
Objective lens: XLPLN10XSVMP (NA 0.6)
Laser: 960 nm (YFP)
Image size: 512 x 512, 170 tiles, Z=8,000 μm, Z Step 16 μm

　

ScaleS
Clearing Mouse Brain Slices and Observing Vasculature with Fluorescently Labeled Lectin
00593_img14.png

Species: Mouse (C57BL6/J, 10.5 days old)
Tissue: Coronal section of cerebral cortex (1.2 mm thick)
Microscope: Confocal microscope (Upright)
Lectin: Texas Red labeled tomato-lectin

　

ScaleA2
Clearing the Whole Brain of YFP-H Mice

Species: Mouse
Tissue: Whole brain
Objective lens: XLSLPLN25XSVMP (NA 0.90, WD 8 mm)

ScaleA2
Clearing the Hippocampus of YFP-H Mice

Species: Mouse
Tissue: Hippocampus
Objective lens: XLPLN25XSVMP (NA1.0, WD 4 mm)

ChemScale
Propidium Iodide (PI) Staining and Clearing of Brain Hemisphere Sections from YFP-H Mice
Propidium Iodide (PI) Staining and Clearing of Brain Hemisphere Sections from YFP-H Mice

Species: Mouse (Thy1-YFP-H line, 42W, Male)
Tissue: Coronal section of the Brain (2 mm thick)
Microscope: Confocal microscope (Evident, FV3000, Inverted)
Objective lens: UPLSAPO10x2 (NA 0.40)
Laser: 488 nm (YFP) / 561 nm (PI)

Protocol (For detailed protocol, see here)

Perfusion fixation + Post-fixation Pretreatment Staining with Fluorescent Dye Clearing Observation
4% PFA/PBS(-) PBS(-) SCALEVIEW-S0 SCALEVIEW-A2 8M Urea SCALEVIEW-A2 descale Solution Fluorescent Dye*/
SCALEVIEW-A2		 SCALEVIEW-A2 descale Solution SCALEVIEW-S4 SCALEVIEW-S4
4°C
3 days		 RT
Several times		 37°C
4 hours		 37°C
4 hours		 37°C
12 hours		 37°C
4 hours		 4°C
6 hours		 RT
2 hours		 RT
2 hours x 1,
1 hour x 1		 4°C
3 hours		 37°C
6-8 hours		 RT
* Examples of fluorescent dyes: 125 nM SYTO™61 (Thermo Fisher Scientific), 500 nM DAPI, 1 μM PI
Volume of Each Solution Used (Approximate volumes for 1-2 mm thick mouse brain hemisphere sections)
[Pretreatment]
SCALEVIEW-S0: 10 ｍL / SCALEVIEW-A2: 10 mL x 2 / descale Solution: 10 mL
[Staining]
Staining solution : 8 mL /descale Solution: 10 mL
[Clearing]
SCALEVIEW-S4: 10 mL
[Observation]
SCALEVIEW-S4: 10 mL
AbScale
Immunostaining and Clearing of Brain Sections from Alzheimer’s Disease Model Mice

Species: Mouse (Alzheimer’s disease model, 17 months old)
Tissue: Brain section (2 mm thick)
Microscope: Confocal microscope (Evident, FV1200, Inverted)
Objective lens: XLPLN10XSVMP (NA 0.60)

Protocol (For detailed protocol, see here)

Perfusion fixation + Post-fixation Pretreatment Immunostaining Clearing Observation
4% PFA/PBS(-) PBS(-) SCALEVIEW-S0 SCALEVIEW-A2 8M Urea SCALEVIEW-A2 descale Solution 1% Blocking agents* /PBS(-) Antibody /AbScale Solution** AbScale Solution** 4% PFA /PBS(-) descale Solution SCALEVIEW-S4 SCALEVIEW-S4
4°C
3 days		 RT
Several times		 37°C
4 hours		 37°C
4 hours		 37°C
12 hours		 37°C
4 hours		 4°C
6 hours		 RT
2 hours		 37°C
> 1 day		 RT
2 hours x 1,
1 hour x 1		 4°C
6 hours		 4°C
6 hours		 37°C
6-8 hours		 RT
* Recommended reagent: Roche, Product Number: 11096176001
** AbScale Solution: 0.33 M Urea, 0.1% (wt/vol) Triton X-100 in PBS
Volume of Each Solution Used (Approximate volumes for 1-2 mm thick mouse brain hemisphere sections)
[Pretreatment]
SCALEVIEW-S0: 10 ｍL / SCALEVIEW-A2: 10 mLx2 / descale Solution: 10 mL
[Immunostaining]
AbScale Solution: 1.5 mL /descale Solution: 10 mL
[Clearing]
SCALEVIEW-S4: 10 mL
[Observation]
SCALEVIEW-S4: 10 mL

　

AbScale-G
Immunostaining and Clearing of Brain Sections from Alzheimer’s Disease Model Mice

Species: Mouse (Alzheimer’s disease model, 60 weeks old)
Tissue: Brain Section (1 mm thick)
Microscope: Confocal microscope (Evident, FV1200, Inverted)
Objective lens: XLPLN10XSVMP (NA 0.60)

Data by courtesy of
Dr. Hama of Laboratory for Cell Function Dynamics, RIKEN Center for Brain Science

AbScale-G improves the permeability of fluorescent dyes, such as Cy3, which were difficult to use with the conventional AbScale method, enabling the use of a broader range of fluorescent dyes and antibodies.

Protocol

Perfusion fixation + Post-fixation Pretreatment Immunostaining Clearing Observation
4% PFA/
PBS(-)		 PBS(-) SCALEVIEW-S0 SCALEVIEW-A2 8M Urea SCALEVIEW-A2 descale Solution Antibody /AbScale-G Solution** AbScale-O
Solution**		 4% PFA /PBS(-) SCALEVIEW-S4 SCALEVIEW-S4
4°C
3 days		 RT
Several times		 37°C
6 hours		 37°C
6 hours		 37°C
6 hours		 37°C
3 hours		 RT
6 hours		 37°C
3 days		 RT
5 minutes x 3 times		 RT
30 minutes		 37°C
6 hours		 RT
* Recommended reagent: Roche, Product Number: 11096176001
** AbScale-O Solution: 0.33 M Urea、0.1% (wt/vol) Triton X-100 in PBS
Volume of Each Solution Used (Approximate volumes for 1 mm thick mouse brain hemisphere sections)
[Pretreatment]
SCALEVIEW-S0: 6 ｍL / SCALEVIEW-A2: 6 mLx2 / descale Solution: 10 mL
[Immunostaining]
AbScale Solution: 1.5 mL /descale Solution: 10 mL
[Clearing]
SCALEVIEW-S4: 8 mL
[Observation]
SCALEVIEW-S4: 8 mL

　

Organoids/Spheroids

AbScale
Immunostaining and Clearing of Neurospheres Derived from Rat Hippocampal Neural Stem Cells

Sample: Neurospheres derived from rat hippocampal neural stem cells
Microscope: Confocal Microscope (Evident, FV1000)
Objective lens: UMPLFLN10XW (NA 0.3)

Protocol

Perfusion fixation + Post-fixation Pretreatment Immunostaining Clearing Observation
4% PFA/
PBS(-)		 PBS(-) SCALEVIEW-S0 SCALEVIEW-A2 8M Urea SCALEVIEW-A2 descale Solution 1% Blocking agents* /PBS(-) Antibody /AbScale Solution** AbScale Solution** 4% PFA /PBS(-) descale Solution SCALEVIEW-S4 SCALEVIEW-S4
4°C
3 days		 RT
Several times		 37°C
4 hours		 37°C
4 hours		 37°C
12 hours		 37°C
4 hours		 4°C
6 hours		 RT
2 hours		 37°C
> 1 day		 RT
2 hours x 1, 1 hour x 1		 4°C
1 hour		 4°C
3 hours		 37°C
4 hours		 RT
* Recommended reagent: Roche, Product Number: 11096176001
** AbScale Solution: 0.33 M Urea、0.1% (wt/vol) Triton X-100 in PBS
Volume of Each Solution Used
[Pretreatment]
SCALEVIEW-S0: 1.2 ｍL / SCALEVIEW-A2: 1.2 mL / descale Solution: 1.2 mL
[Immunostaining]
Follows the AbScale protocol
[Clearing]
SCALEVIEW-S4: 1.2 mL
[Observation]
SCALEVIEW-S4: 1.2 mL
AbScale
Immunostaining and Clearing of Human iPS Cell-Derived Hepatocytes (iCell® Hepatocytes 2.0)
Bright field
FITC
FITC
明視野+FITC
Bright field+FITC

Sample: iCell® Hepatocytes2.0
Plate for spheroid formation: Prime Surface® 96 Slit-well Plate
Primary Antibody: Anti-ABCB11 antibody
Secondary Antibody: Anti-rabbit IgG(H+L), Goat, FITC-conjugated

Protocol

iCell® Hepatocytes 2.0 were matured in 2D culture.
Spheroids were generated using spheroid culture plates.
Clearing was performed with SCALEVIEW

SCALEVIEW Q&A

About protocol

Do samples expand during clearing?
There is minimal change; however, samples may expand slightly during the clearing process. They return to nearly their original size in the final clearing step.
Are there any steps in the clearing process where the procedure can be put on hold?
[Pre-treatment steps for AbScale, AbScale-G, and ChemScale]
During SCALEVIEW-S0 or A2 processing, samples can be kept at 4°C for 12 hours to 3 days.
How should samples be stored after clearing?
Store the samples in SCALEVIEW-S4 solution, sealed by Parafilm to prevent drying, at room temperature and in the dark.

About observation

How long is the fluorescent protein signal retained?
The signal remains stable for approximately six months.
What type of observation container should be used?
Use a dish with a diameter slightly larger than the tissue sample or a See Through Chamber appropriate for the sample’s thickness.
What type of microscope should be used?

Use a confocal or two-photon microscope. Ensure that the objective lens is compatible with the refractive index of SCALEVIEW-S4 (RI=1.47) or SCALEVIEW-SMt (RI=1.49).

Tips: If a compatible microscope is unavailable or observation under high refractive index conditions is difficult, consider using SCALEVIEW-A2 (RI=1.38) as a substitute. For more details, refer to the information provided.

References

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See Through Chamber

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