Tissue Clearing and 3D Staining Kits

Technical Guidebook

1

INDEX

01

Technical Overview

302

CUBIC Tissue Clearing Reagents

4

Application Example 1: Tissue Transparency of Organs

5(Reagents used: CUBIC-L, CUBIC-R)

7

Application Example 2: Clearing Organ Tissues Including Bones（Reagents used: CUBIC-L, CUBIC-R, CUBIC-B）

03

8

CUBIC-HV™2 3D Tissue Staining Kit
Application Example 3: Uniform Staining of 3D Sample

9（Kit used: CUBIC-HV™2 kit）

04

FAQs and References

11

2

01

Technical Overview
Our CUBIC Technology was developed by RIKEN and the University of Tokyo over about 10

years to allow observation of all cells in organs and the body. Today, our technology isused by researchers around the world and gained high trust as a versatile tissue clearing

and 3D tissue staining method. Our kits are designed for safe use and easy introduction.

CUBIC Tissue Clearing Reagent Kits
 The world’s most powerful and highly versatile water-soluble clearing reagents that allow
transparency of organs and the whole body.
 Can be performed with only general lab equipment and does not require special dedicatedequipment such as an electrophoresis apparatus.
 Uses light-sheet microscopes (LSFM) and confocal laser scanning microscopes to allow
imaging of the whole 3D organ with a resolution to the cellular level.

Tissue Transparency Workflow

PFA fixed sample

Remove lipids
and pigmentswith CUBIC-L

RI matching with
CUBIC-R

Transparency
sampleCUBIC-HV 3D Staining Kit
 The world’s highest-performance 3D staining technology (patented by CUBICStars, Inc.) Practical application of a special tissue staining process that uniformly stains 3D blocks oftissue Highly reproducible protocol compatible for various nuclear staining and antibody staining

Main Technology
Patented Technology 1

Patented Technology 2

High concentration
stain probe method

Penetration enhancer
stain probe

A new technology that allows
concentrationlevelstoincrease 10 to 100 times thantheregularmethod,penetrating
reagents
andantibodies to the depths
quickly.

An additive that penetrates
reagents and antibodies tothe depths quickly made from
ideal compounds selectedamong over 500 types.

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02

Tissue Clearing Reagent Kit CUBIC

Features Highly versatile water-soluble CUBIC tissue clearing kitsthat have a powerful transparency level Compatible with all organs including the bone. Fluorescentprotein signals can be observed after clearing the sample.

 Our high simplicity and reproducibility protocol solves

issues pertaining to safety, waste processing, microscopecompatibility, fluorescent protein signal retainability, etc.

Products
CUBIC Basic Clearing Reagents
Allows animal organs to become transparent by immersing in two types of reagents
USD580/kit

CUBIC-L [CSCR001]

For delipidation and decolorization

CUBIC-R [CSCR002]

For clearing (RI matching). High versatility andeasy handling.

500 mL/kit
USD 400 /kit
500 mL/kit*Can experiment approx. 20 mouse brains with 500 mL of reagent.
*CUBIC-R causes each axis to swell 1.5 times to increase transparency and performance resolution during microscope imaging.
*The product structure of kits is subject to change without prior notice.

CUBIC Bone Tissue Clearing Reagent

Reagent optimized for bone tissue transparency. Use with CUBIC-L/R.
CUBIC-B [CSCR003]

Bone tissue clearing reagent optimized for bone
tissue decalcification

USD 165 /kit
100 mL/kit*Can experiment approx. 5 to 10 mouse lower limbs with 100 mL of reagent.
*The product structure of kits is subject to change without prior notice.
Reference 1 K. Matsumoto, H. R. Ueda, et al., Nat. Protoc. 2019, 14, 3506-3537
Reference 2 K. Tainaka, H. R. Ueda, et al., Cell Rep. 2018, 24, 2196-2210

https://doi.org/10.1038/s41596-019-0240-9
https://doi.org/10.1016/j.celrep.2018.07.056

402

CUBIC Tissue Clearing Reagent Kit : Application Example 1

Basic Process of Clearing and Staining Mouse Organs
Around 7days

3 - 5 days
Fixation*1

CUBIC-L
Delipidation and

CUBIC-HVTM2CUBIC-R

3D Staining

RI Matching

Decoloration

Wash
Preserve

d

Wash

Wash

2 hrs×3

2 hrs×3

Postfixation
24 hrsSamples can be preserved*The number of days is based onwhen using adult mouse brains.

Wash

2 hrs×3

RepeatClearing*Note 3Samples can be preservedin preservative-added PBS

in preservative-added PBS

at 4℃

at 4℃ *Note 2*Note 1 We recommend using 4% paraformaldehyde (pH 7~7.5) for the fixative. Transparency is greatly affected by fixation conditions. It is important to minimize

the difference between controlled conditions, such as creating large amounts of paraformaldehyde and storing them in the freezer. Overfixation andyellowing may occur if the pH is alkaline.

*Note 2 Transparency samples can be embedded with agarose gel. This allows stable preservation for a long time.
*Note 3 Frequently repeating clearing results in tissue damage.

Mouse Organ Clearing Procedure (Basic Protocol)
Step

Reagent

Temperature

Time

Extract
organ

Notes
After perfusion fixation.

Postfixation

4% PFA in PBS

4℃

1 day

Wash x 3

PBS

RT

Up to 2 hrs x 3

Beforereplacement

50% CUBIC-L

6 ‒ 24 hrs

Optional step. Mix an equal amount of CUBIC-L and water.

Delipidation
anddecolorationRT
Or 37℃CUBIC-L

37℃> 2 days

Change CUBIC-L to a new solution every two days.

Wash x 3

PBS

RT

Up to 2 hrs x 3

Staining

CUBIC-HVTM2, stain
probe (antibody,staining)

Exchange or wash the tube every time to prevent carry-over ofthe solution.

RT

Up to 7 days

Optional step.

Wash x 3

CUBIC-HVTM24℃

Up to 1.5 hrs x
3Conduct if staining.

Post-fixation

1% formaldehyde

4℃

1 day

Conduct if staining. Use 37% formaldehyde diluted with PBS.

Post-fixation

1% formaldehyde

37℃

1 hr

Conduct if staining. Transfer the solution treated overnight at
4℃ to the solution treated at 37℃.

Wash x 3

PBS

RT

Up to 2 hrs x 3

Beforereplacement

50% CUBIC-R

RT
Or 37℃

1 day

Clearing

CUBIC-RRT
Or 37℃

Shake gently (the same applies to the steps below).

Conduct if staining.
Mix an equal amount of CUBIC-R and water.

> 1 day*Details on the 3D staining procedure on pages 8 – 10.

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CUBIC Tissue Clearing Reagent Kit : Application Example 1

Practical Example of Mouse Organ Clearing

Adult mouse brain after extraction and PFA
fixation

After replacement with 50% CUBIC-L(Treated overnight at 37℃)

After delipidation and decoloration with
100% CUBIC-L(Treated for four days at 37℃)

After washing with PBS

After shaking in 4 mL 50% CUBIC-R(Treated overnight at room temperature)

After shaking in 4 mL CUBIC-R (overnight at room
temperature),

Immersed in mounting agent for observation (RI = 1.522)* Amount of reagent and treatment time varies depending on the organ.
* Please use a slightly larger tube wider than the organ’s diameter. Most of the organ should be immersed in the reagent when the tube islaid on its side.
* Shake the organ immersed in the reagent at a speed that shakes the whole organ.
* We do not recommend rotation as it will create bubbles.

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CUBIC Tissue Clearing Reagent Kit : Application Example 2

Mouse Bone Tissue Clearing Basic Process

CUBIC-L

Fixation

Delipidation andDecoloration

Decalcification

Delipidation and

CUBIC-HVTM2CUBIC-R

3D Staining

RI Matching

Decoloration

Wash

2 hrs×3

2 hrs×3*Time is based when conductedon adult mouse lower limb.

CUBIC-L

CUBIC-B

Wash

Wash
Preserve

Around 7days

3 - 5 days

5 – 7 days

3 – 5 days

Wash

Wash

2 hrs×3

Samples can be preserved

Samples can be preserved in preservative-added PBS at 4℃.

Mouse spinal

RepeatClearing

2 hrs×3

in preservative-added PBS
at 4℃.

Mouse spinal

cord before

cord after

clearing

clearing

Mouse Bone Clearing Procedure (Basic Protocol)
Step

Reagent

Temperature

Time

Fixation

4% PFA in PBS

4℃

1 day

Prior
replacement

50% CUBIC-L

RT
Or 37℃

6 ‒ 24 hrs

Optional step. Mix an equal amount of CUBIC-L and water.

Delipidation

CUBIC-L

37℃

3 - 7 days*

Shake gently (the same applies to the steps below). Change
once every two days.

Wash

PBS

RT

Up to 2 hrs x3

Exchange or wash the tube every time to prevent carry-over ofthe solution.

Decalcification

CUBIC-B

37℃

5 - 7 days

Wash

PBS

RT

Up to 2 hrs x3

Delipidation

CUBIC-L

37℃

2 - 4 days

Wash

PBS

RT

Up to 2 hrs x3

Stain

CUBIC-HVTM2, staining
probe

RT

5 - 7 days

Wash x 3

CUBIC-HVTM24℃

Up to 1.5 hrs x
3

Post-fixation

1% FA

4℃

1 day

Conduct if staining. Use 37% formaldehyde diluted with PBS.
Conduct if staining. Transfer the solution treated overnight at
4℃ to the solution treated at 37℃.

Post-fixation

1% FA

37℃

1 hr

Wash x 3

PBS

RT

Up to 2 hrs x 3

Beforereplacement

50% CUBIC-R

Clearing

CUBIC-RRT
Or 37℃
RT
Or 37℃

1 day

Notes

Change CUBIC-L to a new solution at least once.
Exchange or wash the tube every time to prevent carry-over ofthe solution.
Exchange or wash the tube every time to prevent carry-over ofthe solution.
Optional step.
Conduct if staining.

Conduct if staining.
Mix an equal amount of CUBIC-R and water.

> 1 day
Reference

Tainaka et al. Cell Reports 2018

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CUBIC-HV™2 3D Tissue Staining Kit

Features Productized based on CUBIC-HistoVIsion, the world’s
highest-performance 3D tissue staining technology.
 The kit includes reagents that allow equal staining of
the inside and outside of the 3D samples.
 To be used together with CUBIC clearing reagents (soldseparately).
 Available in a starter kit that includes a staining pot
optimized for experiments and a general kit (the staining
pot can be used repeatedly).

Products
Buffers and tools optimized for 3D tissue staining. Applicable for antibody and nuclear staining.
Equally stains a whole mouse brain within a week at the shortest.

Product Name

Kit ContentsPrice Pre-wash buffer Staining buffer

CUBIC-HV™2 3D tissue
staining kit [CSSR003]

 Additive A - F

USD 990/kit

 Wrapping reagent

10 tests/kit

 Wash buffer Supplement for staining buffer (supplement)

CUBIC-HV™2 3D tissue
staining kit (Starter) The above reagents ＋ Staining pot[CSSR004]

3D tissue staining pot
for CUBIC-HV™2[CSSR005]

 Staining pot

USD1,580/kit
10 tests/kit

USD 590/kit
10 tests/kit*The amount used in the above structure is applicable in the application example of adult mouse brains. The amount required will varydepending on the size and type of organ.
*The product structure of kits is subject to change without prior notice.
*Dissolve the supplement in the staining buffer right before use. Dissolving takes approx. 2 hours.
*Purchase formaldehyde available in the market separately.

Reference E. A. Susaki, H. R. Ueda, et al., Nat. Commun. 2020, 11, 1982. https://doi.org/10.1038/s41467-020-15906-5

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CUBIC-HV™2 3D Staining Kit : Application Example 3

Basic Process of 3D Staining Mouse Brains Using CUBIC-HVTM2CUBIC-HVTM 2 Kit
3D Staining
3 – 5 days
CUBIC-L

Fixation

1 day

1.5 hrs

6 – 7 days

1 day

PrewashStaining
bufferreplacement

Nuclearstaining
Immunostaining

4℃
Reaction

Delipidation
andDecoloration

CUBIC-R
RI Matching

Wash

Postfixation

Wash

Wash
Preserve

1.5 hrs
×３

2 hrs×3

24 hrsSamples can be preserved

Wash

RepeatClearing

2 hrs×3

Samples can be preservedin preservative-added PBS*The number of days is based
at 4℃.
on when using adult mouse brains.

in preservative-added PBS
at 4℃.

Steps to 3D Staining Mouse Brains (Basic Protocol)
Step

Reagent

Temperature

Time

Extract organ

Notes
After perfusion fixation.

Post-fixation

4% PFA in PBS

4℃

1 day

Wash x 3

PBS

RT

Up to 2 hrs x 3

50% CUBIC-L

RT
Or 37℃

6 ‒ 24 hrs

Optional step. Mix an equal amount of CUBIC-L and water.

CUBIC-L

37℃> 2 days

Change CUBIC-L to a new solution every two days.

Wash x 3

PBS

RT

Up to 2 hrs x 3

Staining
pre-wash

1× CUBIC-HV™2
Pre wash buffer

37℃

Overnight

Beforereplacement
Delipidation
andDecoloration

Shake gently (the same applies to the steps below).

Exchange or wash the tube every time to prevent carry-over ofthe solution.

Staining before
replacement

2× CUBIC-HV™2
Staining buffer

37℃

1.5 hrs

Staining

2× CUBIC-HV™2
Staining buffer

Add the additive.
Conduct reactions of the primary antibody and secondary antibody (Fabfragment) separately during replacement.

RT~37℃

6 - 7 days

Add the sample, additive, antibody solution, and nuclear staining reagent
to the staining pot. Conduct staining in the wrapping reagent.

2× CUBIC-HV™2
Staining buffer

4℃

Overnight

Set the reaction chamber in the previous step to 4℃ as it is.

Wash x 3

2× CUBIC-HV™2Wash buffer

4℃

Up to 1.5 hrs x
3

Post-fixation

1% Formaldehyde

4℃

1 day

Use 37% formaldehyde diluted with PBS.

Post-fixation

1% Formaldehyde

37℃

1 hr

Transfer the solution treated overnight at 4℃ to the solution
treated at 37℃.

Wash x 3

PBS

RT

Up to 2 hrs x 3

Beforereplacement

50% CUBIC-R

RT
Or 37℃

1 day

Clearing

CUBIC-R

Reactionafter staining

RT
Or 37℃Store the buffer in ice beforehand.

Mix an equal amount of CUBIC-L and water.

> 1 day9

03

CUBIC-HV™2 3D Staining Kit : Application Example 3

3D Staining Mouse Organ Practical Example

Adult mouse brain after treating with
CUBIC-L

Pour the solution containing the preservative,
staining probe, and staining buffer into thesample pot.

Add the sample treated with CUBIC-L andsample wrapping reagent for staining.

Set the sample in a rotator. Rotate at a speed thatmaintains a relative top and bottom position inside
the pot without the sample flipping.

*If the sample flips inside the pot, the top and bottom of the sample will

always be upward/downward in the gravity direction, resulting in unequal
staining.

After staining, remove the sample from the pot

and wash with the wash buffer. Shake vigorously
in the first wash to remove the sample wrapping
reagent.

Conduct clearing with CUBIC-R after washingand post-fixation.

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Frequently Asked Questions

Questions on Staining Reagents, Antibodies, and Fluorescent Proteins

Q
A

What fluorescence reagents and stains can be used in
CUBIC-HV™2？

When using antibodies, we recommend using antibodies that are directly fluorescently labeled with
Alexa Fluor® dyes or FabuLightTM Fab fragment secondary antibodies from Jackson Immunolab.

Regarding Alexa Fluor® dyes, AF488 is not compatible with CUBIC-R. Please use dyes such as FITC

instead. In the CUBIC-HV protocol, we do not recommend using general IgG secondary antibodies, as

staining the depths is not guaranteed. For nuclear staining, we recommend propidium iodide (PI),SYTOXTM-Green, or RedDotTM2.

Q
A

Can primary antibodies available in the lab be used with CUBIC-HV™2？
In the references, some proteins that have maintained their antigenicity before and after clearing.
However, not all proteins have been confirmed, so consider the antibodies at hand before use.

To check if the antibodies are compatible, compare the stains of slices before and after processing with
CUBIC-L.

Q
A

Can fluorescently labeled secondary antibodies be used after primary
antibodies, similar to normal immunohistostaining?

For CUBIC-HV™2, the FabuLightTM Fab fragment secondary antibody reacts with the primary
antibody to create a complex before 3D staining. We do not recommend staining 2D slices in
the general two steps (primary antibody to secondary antibody) because staining of thedepths is not guaranteed.

Q

What fluorescent proteins can be used?

A

Fluorescent signals are maintained for EGFP, EYFP, Venus, tdTomato, mCherry, andmKate2. For other fluorescent proteins, please check before use on your own.

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Frequently Asked Questions

Questions on Clearing
Q

What types of containers should be used during clearing?

A

CUBIC tissue clearing is designed to improve transparency and microscope resolution byslightly expanding the tissue. Thus, we recommend using tubes, containers, etc. with adiameter wider than the tissue. For example, when a tube is filled halfway with the reagent,
the sample should not come out of the liquid surface when the tube is laid on its side.

CUBIC reagents are aqueous, so polypropylene and polyethylene containers can be usedsafely.

Q

Will the expansion of tissues affect the experiment?

A

Various cells may expand, but the physical relationship of the cells is maintained. The

rigidness of the structure may vary depending on the tissue (nerves, veins, etc.) and may
cause artifacts, so confirmation is required according to the situation.

Q

Is fixation still required even when clearing is done right after the organ isA

CUBIC tissue clearing agents are optimized for PFA fixation. Lysis will occur on samples thatextracted from the animal?

are not fixed, so please fix the samples.

Q
A

Can samples be cleared even if some time has passed after dissection
and fixation?

Samples that have been immersed in fixative for a long time (over several weeks) can still becleared.

However, since the fixation condition is a parameter that greatly affects the transparency

condition, we recommend maintaining the preparation condition and fixation period of the
fixative as much as possible. In our protocol, we recommend washing with PBS within 24

hours after perfusion fixation of the animal, and promptly starting the CUBIC-L treatment.

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Frequently Asked Questions

Questions on Clearing
Q

Can paraffin-embedded samples become transparent?

A

Paraffin-embedded samples can become transparent after deparaffinization treatment.
Please refer to the following treatment method for more details.

Reference CUBIC pathology: three-dimensional imaging for pathological diagnosis
S. Nojima et al., Sci. Rep. 2017, 7, 9269.

https://doi.org/10.1038/s41598-017-09117-0

Q

How much reagent is required for clearing?

A

When making the whole mouse body transparent, the amount of reagent should beenough to immerse the whole body. For organs, choose a tube so that when the tube isfilled halfway with the reagent, the whole organ is immersed when the tube is laid on its

side. For example, for mouse brains, pour 10 to 15 mL of CUBIC-L into a 20 to 30 mL tube.
The actual amount will vary according to the sample size and container used. As an

example, 200 to 400 mL of CUBIC-L and 100 to 200 mL of CUBIC-R is required to clear

the whole mouse body, and a total of 20 to 40 mL of CUBIC-L and 10 to 20 mL of CUBICR is required to clear each mouse organ (approx. 1cm3).

Q

What is the reason if clearing does not go well?

A

It may be due to the following reasons. Please consider the measures below.
a) The pH level of the PFA solution used in fixation is highWhen the pH level is over 8, overfixation occurs, and the organ becomes yellow,
causing clearing to slow down. Please set the pH level around 7 to 7.5.

In addition, the transparency level differs depending on the PFA pH level andprocessing time. We recommend maintaining the fixation time and fixative
preparation procedure as much as possible.
b) Delipidation is not complete.

Either extend the delipidation step or consider the exchange frequency of CUBIC-L.

We recommend exchanging to a new CUBIC-L solution every two days and shaking at
37℃.

c) Clearing is not complete.

When moisture is added during CUBIC-R, the RI drops, and transparency becomesinsufficient. Please exchange CUBIC-R.

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04

Frequently Asked Questions

Questions on Clearing
QAHow long does delipidation take?
For adult mouse tissue, delipidation takes three to seven days. The length of delipidation
depends on the organ.

Set the delipidation time according to the type and size of the organ, and experiment
purpose. For example, complete transparency is required for light-sheet microscopeobservation, but less time and incomplete transparency are acceptable for partial
observation using two-photon microscopes.

Questions on After Clearing
Q

What should we do with the liquid waste after the experiment?

A

Dispose of the liquid waste according to the specified disposal method after consultingwith the person in charge of waste in your institution. Furthermore, in general, samples
and reagents used in the experiment should be disposed of as medical waste andinfectious waste, and unused CUBIC disposed of as organic waste and fire-resistantwaste because it has a high water content.

QA

How should samples be stored after clearing?
Samples can be stored at room temperature with the used CUBIC-R. Furthermore, thesample may harden if the water in the CUBIC-R solvent evaporates. Seal the container
with parafilm, etc.

Samples can also be stored at room temperature in agarose gel. This method is morestable.

To archive, samples can be stored at 4℃ inside the preservative-added PBS, such as
sodium azide after washing CUBIC-R with PBS.
【Agarose Gel Storing Procedure】

In an appropriate tube, add 2% agarose gel to the CUBIC-R reagent used in the clearing.
Heat and dissolve the solution. Insert the transparent sample into the agarose solution
before it hardens. Cool enough to harden.

For more details, refer to the article or website listed below.
Website for reference:
Reference:

http://cubic.riken.jp/

Advanced CUBIC tissue clearing for whole-organ cell profiling

K. Matsumoto et al., Nat. Protoc. 2019, 14, 3506. https://doi.org/10.1038/s41596-019-0240-9

14

04

Frequently Asked Questions

Questions on After Clearing
Q
A

The transparent sample cannot be observed well.
We recommend observing with light-sheet microscopes for tissue clearing observation(LSFM), confocal laser scanning microscopes (CLSM) with objective lenses compatible
with high RI, and two-photon microscopes. When observing, immerse the sample in themounting agent for observation, and use objective lenses compatible with the RIs. In
recent years, affordable light-sheet microscopy for tissue clearing is developed.
Reference:
Users

descSPIM: Affordable and Easy-to-Build Light-Sheet Microscopy for Tissue Clearing TechniqueK. Otomo et al., bioRxiv. 2023 https://doi.org/10.1101/2023.05.02.539136

Q

What is the refractive index of the reagents?

A

The refractive index (RI) of CUBIC-R is 1.522. Avoid mixing solvents such as water with

CUBIC-R to change the refractive index.

Q

Are CUBIC-L and CUBIC-R the same as CUBIC-1 and CUBIC-2

A

CUBIC-1 (Scal eCUBIC-1, Reagent-1) and CUBIC-2 (Scal eCUBIC-2, Reagent-2) are

mentioned in the articles?

different from CUBIC-L and CUBIC-R. CUBIC-1 and CUBIC-2 are the first-generation

CUBICs, and CUBIC-L and CUBIC-R are the improved second-generation CUBICs withmuch higher transparency.

For functions, CUBIC-1 shows the same delipidation and decoloration as CUBIC-L, and
CUBIC-2 shows the same refractive index matching as CUBIC-R.

*Regarding the information above, clearing and staining results vary depending on the transparency sample, staining
reagents, and devices used.

Please consider the appropriate processing time and reagent concentration level.

15

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References

Transparency Examples of the Clearing of the Mouse Body and Organs (Brain, Lungs, Liver, Limb,
and Kidney), Marmoset Brain, and Human Organs (Brain, Kidney, Liver, and Lungs)(Immunostaining Protocol After Clearing with CUBIC)

Chemical Landscape for Tissue Clearing based on Hydrophilic ReagentsK. Tainaka, T. C. Murakami, E. A. Susaki, C. Shimizu, R. Saito, K. Takahashi, A. Hayashi-Takagi, H. Sekiya, Y. Arima, S. Nojima, M.

Ikemura, T. Ushiku, Y. Shimizu, M. Murakami, K. F. Tanaka, M. Iino, H. Kasai, T. Sasaoka, K. Kobayashi, K. Miyazono, E. Morii, T. Isa,
M. Fukayama, A. Kakita, H. R. Ueda, Cell Rep. 2018, 24, 2196. https://doi.org/10.1016/j.celrep.2018.07.056

Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution

S. I. Kubota, K. Takahashi, J. Mishida, Y. Morishita, S. Ehata, K. Tainaka, K. Miyazono, H. R. Ueda, Cell Rep. 2017, 20, 236.
http://doi.org/10.1016/j.celrep.2017.06.010
Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis

E. A. Susaki, K. Tainaka, D. Perrin, F. Kishino, T. Tawara, T. M. Watanabe, C. Yokoyama, H. Onoe, M. Eguchi, S. Yamaguchi, T. Abe, H.

Kiyonari, Y. Shimizu, A. Miyawaki, H. Yokota, H. R. Ueda, Cell 2014, 157, 726. http://doi.org/10.1016/j.cell.2014.03.042
Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

K. Tainaka, S. I. Kubota, T. Q. Suyama, E. A. Susaki, D. Perrin, M. Ukai-Tadenuma, H. Ukai, H. R. Ueda, Cell 2014, 159, 911.
http://doi.org/10.1016/j.cell.2014.10.034

Application Example of CUBIC for Human Pathological Diagnosis
CUBIC pathology: three-dimensional imaging for pathological diagnosis

S. Nojima, E. A. Susaki, K. Yoshida, H. Takemoto, N. Tsujimura, S. Iijima, K. Takachi, Y. Nakahara, S. Tahara, K. Ohshima, M.

Kurashige, Y. Hori, N. Wada, J. Ikeda, A. Kumanogoh, E. Morii, H. R. Ueda, Sci. Rep. 2017, 7, 9269. https://doi.org/10.1038/s41598-017-

09117-0

Whole Organ/Body 3D Staining and Observation with CUBIC-HistoVIsion (CUBIC-HV™ Kit Basic
Protocol)Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues

E. A. Susaki, C. Shimizu, A. Kuno, K. Tainaka, X. Li, K. Nishi, K. Morishima, H. Ono, K. L. Ode, Y. Saeki, K. Miyamichi, K. Isa, C.

Yokoyama, H. Kitaura, M. Ikemura, T. Ushiku, Y. Shimizu, T. Saito, T. C. Saido, M. Fukayama, H. Onoe, K. Touhara, T. Isa, A. Kakita, M.
Shibayama, H. R. Ueda, Nat. Commun. 2020, 11, 1982. https://doi.org/10.1038/s41467-020-15906-5

Affordable Light-Sheet Microscopy for Tissue Clearing Observation

descSPIM: Affordable and Easy-to-Build Light-Sheet Microscopy for Tissue Clearing Technique Users

K. Otomo, T. Omura, Y. Nozawa, Y. Saito, E. A. Susaki

bioRxiv 2023. https://doi.org/10.1101/2023.05.02.539136

Protocol Article on Tissue Clearing and Nuclear Staining
Advanced CUBIC tissue clearing for whole-organ cell profiling

Katsuhiko Matsumoto, Tomoki T. Mitani, Shuhei A. Horiguchi, Junichi Kaneshiro, Tatsuya C. Murakami, Tomoyuki Mano, Hiroshi

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Nature Protoc. 2019 https://doi.org/10.1038/s41596-019-0240-916

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