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Animal Tissue-Clearing Reagents"CUBIC"

Tissue-Clearing Reagent
 A tissue clearing method "CUBIC" has been developed by Prof. Hiroki R. Ueda and coworkers at The University of Tokyo / RIKEN. The CUBIC technique enables cyclopedic imaging at a single-cell resolution following whole-body and whole-organ clearing. CUBIC reagents that can be used in the tissue clearing method are provided by TCI.

*CUBIC reagents are commercialized under invention licenses by RIKEN, Japan.

Both of CUBIC-L (Product Number: T3740) and CUBIC-R+ (Product Number: T3741) are required for tissue-clearing.
Please use a combination with CUBIC reagents according to your samples and purposes.

Advantages

BASIC PROTOCOL: Soaking in only two products makes mouse whole-body or animal tissues clear. CUBIC-L (for delipidation and decoloring) and CUBIC-R+ (for RI matching).

CUBIC-L: for delipidation and decoloring
CUBIC-R+: for RI matching

OPTIONAL PROTOCOL: The following products can easily clear tissues which were previously difficult to clear.

CUBIC-B: for bone
CUBIC-HL: for highly fatty tissues and quenching autofluorescence
CUBIC-P: for mouse perfusion efficiently aids with perfusion fixation

EXPANSION PROTOCOL: The following products can clear tissues with expansion.

CUBIC-X1: for expansion tissues
CUBIC-X2: for RI matching with keeping the expanded size

Preserve the fluorescent protein signals except CUBIC-HL.


Shorter sample treatment period.


Using light-sheet fluorescent microscopy (LSFM) or confocal laser-scanning microscopy (CLSM) enables the whole-organ/body imaging at a cellular resolution.


Example : Mouse whole-body clearing

Whole-body clearing of mouse by CUBIC

Figure 1. Whole-body clearing (Left), Whole-body clearing with propidium iodide staining (Right)

Mouse whole-body clearing procedure
Mouse whole-body clearing procedure
Mouse whole-body clearing procedure for staining
1) nuclear staining by propidium iodide (PI)
Mouse whole-body clearing procedure for nuclear staining by propidium iodide (PI)
2) nuclear staining by RedDot2
Mouse whole-body clearing procedure for nuclear staining by RedDot2

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Example : Mouse whole-organ clearing

Whole-organ clearing of mouse by CUBIC

Figure 2. Whole-brain clearing (Left),
Whole-brain clearing with RedDot 2 staining and immunostaining (Right)

Mouse whole-organ clearing procedure
>Mouse whole-organ clearing procedure
Mouse whole-organ clearing procedure for staining
Example : immunostaining
Mouse whole-organ clearing procedure for staining

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Example : The efficient clearing of adult mouse (more than 6-week-old) whole-body or organ samples

The efficient clearing procedure of adult mouse (more than 6-week-old) whole-body or organ samples
The efficient clearing of adult mouse (more than 6-week-old) whole-body or organ samples

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Example : The clearing of mouse body or tissues including bone

Mouse body or tissues including bone clearing by CUBIC
The clearing procedure of mouse body or tissues including bone
The clearing procedure of Mouse body or tissues including bone

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Example : The clearing of human brain tissue large blocks

The clearing of human brain tissue large blocks by CUBIC
The clearing procedure of human brain tissue large blocks

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Example : The clearing of aggressive human tissue

The clearing procedure of human brain tissue large blocks by CUBIC
The clearing procedure of aggressive human tissue
The clearing procedure of aggressive human tissue

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Q&A

===========================>>[Click here to view Q&As]<<===========================

About Staining Reagents

Q: What kind of staining reagents can be used?

A: a) Antibodies: direct fluorescent-labeled antibodies are preferred. For example, the antibodies are diluted adequately in PBS containing both 0.5% Triton X-100 and 0.01% NaN3.
  b) Nuclei staining reagents: propidium iodide can be used. Propidium iodide is diluted to 10 µg/mL with 0.1 M phosphate buffer (pH 7.4), containing 0.5 M NaCl.


Q: Can antibodies from my laboratory be used?

A: Some proteins do not change their antigenicity during fixation or tissue-clearing procedures. However, this is not conformed for all the proteins. It is recommended to initially test the antibodies you are already using.


Q: Can fluorescent-labeled secondary antibodies be used?

A: We do not have information about protocols using secondary antibodies. It takes significant amounts of time for the two-step treatment with both primary and secondary antibodies. Therefore, we recommend directly labeling your primary antibody with fluorescent reagents.


Q: What kind of fluorescent proteins can be used?

A: We assessed the retention of fluorescence intensity in GFP. Some fluorescent proteins, such as, EGFP, EYFP, mCherry, and mKate2, have been confirmed to retain their fluorescence signals (Cell 2014, 157, 726-739.)

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During Clearing Steps

Q: What kind of container is suitable?

A: Any container that is slightly larger than the organism specimen being cleared is suitable. For mouse organ clearing, a container that is slightly bigger than the organ itself is recommended because the organ may expand during the clearing procedure. CUBIC products are aqueous-based reagents, thus they can be used safely with any laboratory plasticware such as polypropylene or polyethylene.


Q: Does tissue swell? If yes, will this influence the experiment in any way?

A: The tissue or organs may expand during the clearing; However, it has been reported that relative cell position remains the same. Thus, expansion is linear and uniform.


Q: Could the sample fixation step be omitted because the organs undergo the clearing steps as soon as they are excised?

A: In the samples without fixation, the structure of cells may be destroyed, thus, they should be fixed before clearing steps.


Q: Is it possible to clear the samples after they were excised and fixed?

A: The samples which were soaked in fixing solution for several weeks or which are stored at -80°C for several months after fixation can still get cleared.


Q: What quantity of these reagents are required for tissue clearing?

A: For mouse whole-body clearing, the volume of reagents used must be sufficient to submerge the entire specimen. For example, for the whole-body clearing of a mouse, 200 to 400 mL of CUBIC-L and 100 to 200 mL of CUBIC-R+ are needed. For mouse tissue clearing, the volume of reagents needed is half the volume of the organ being cleared. For example, 20 to 40 mL of CUBIC-L and 10 to 20 mL CUBIC-R+ are needed.


Q: The clearing of organs or bodies was interrupted and did not occur successfully.

A: There are a few possible reasons. Consider the following troubleshooting options.

a) The pH of PFA solution for fixing organs or bodies is too high.
When the pH is more than 8, organs and bodies become over-fixed and are less cleared, thus, the pH should be adjusted between 7 – 7.5.
b) Delipidation is incomplete.
Samples are immersed in CUBIC-L with gentle shaking at 37 °C for 2 – 5 days or more, and fresh CUBIC-L should be used daily.
c) Clearing is incomplete.
The clearing time period can be extended. Additionally, consider replacing and using a fresh CUBIC-R+ solution.

Q: How long does it take to delipidate samples?

A: Approximately 3 days are required to delipidate the lung, intestine, pancreas and spleen of an adult mouse, and approximately 5 days to delipidate the heart, brain, liver and kidney.

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After Clearing Samples

Q: How should the reagents be disposed of following use?

A: Please dispose of the reagents according to the regulations of your institution. Reagents used to soak animal or organ samples are typically treated as medical waste. The unused CUBIC-L, and R+ reagents are non-flammable waste liquids. Please refer to the included package insert for reagent descriptions and constituencies.


Q: How should clearing samples be stored?

A: Clearing samples can be stored at room temperature in CUBIC-R+ or CUBIC-X2. CUBIC-R+ and CUBIC-X2 contain many solutes and a little water as a solvent. Thus, the samples should be stored sealed by parafilm or other means to prevent reagents from evaporation. The agarose gel embedding sample can also be stored at room temperature.


[How to embed in agarose gel]

Add agarose powder to the used CUBIC-R+ to a final concentration of 2%(w/v) in a tube, and dissolve it by heat. Embed samples into the mixture before gelation, and prepare the gel by cooling it. This agarose gel can be stored at room temperature, and if required, the head of the tube can be cut and the gel pushed out. The surface of the gel becomes dry and white when it is pushed out; therefore, it should be used immediately after pushing.

Q: Clearing samples cannot be observed well.

A: Light-sheet fluorescent microscopy (LSFM) or confocal laser-scanning microscopy (CLSM) is recommended for the observation of the samples. Clearing samples become gel-like and it may be difficult to cut them into thin slices. For microscopic observation, embed samples in agarose gel, cut them out into an appropriate shape, and then observe them use.


Q: What is the refractive index (RI) of CUBIC reagents?

A: The RI of CUBIC-R+ is 1.52 and that of CUBIC-X2 is 1.467. The objective lens or the immersion oils which are suitable for these RIs should be used. They should not be mixed with other solvents such as water in order to change their RIs.


Q: CUBIC-1, CUBIC-2 have been described in some papers, are they the same as CUBIC-L, CUBIC-R+?

A: CUBIC-1, CUBIC-2 differ from CUBIC-L, CUBIC-R+ in terms of their clearing ability, as CUBIC-L, CUBIC-R+ is superior. CUBIC-1 and CUBIC-L play the same role in delipidation and decoloring, and CUBIC-2 and CUBIC-R+ play the same role in RI matching. CUBIC-R also differs from CUBIC-R+. CUBIC-R is composed of nicotinamide and CUBIC-R+ is composed of N-methylnicotinamide. CUBIC-R+ is superior to CUBIC-R in terms of maintaining fluorescent signals.


**The clearing or staining result differ according to the samples or staining reagents. Please examine the treatment time or the concentration of staining reagents.

References


Using CUBIC-L, CUBIC-R+, CUBIC-B, CUBIC-HL, CUBIC-P,
Mouse Whole Body, Brain, Lung, Liver, Leg, Kidney, Marmoset Brain, Human Brain, Kidney, Liver, Lung Clearing
[Immunohistochemistry after CUBIC protocol]


Mouse Whole Body, Brain, Lung Clearing


Mouse Brain, Marmoset Brain Clearing


With CUBIC Perfusion,
Mouse Whole Body, Heart, Lung, Kidney, Liver Clearing


Using CUBIC-X1 and CUBIC-X2,
Mouse Brain Expansion

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