Enabling 3D Observation of Tissues through Clearing Tissue Clearing Reagent Kit CUBIC

A1.When using antibodies, we recommend using antibodies that are directly fluorescently labeled with Alexa Fluor® dyes or FabuLight™ 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), SYTOX™-Green, or RedDot™2

A2. In the references, some proteins have maintained their antigenicity before and after clearing. However, since not all proteins have been confirmed, please consider the antibodies at hand before use. To check if the antibodies are compatible, please compare the stains of slices before and after processing with CUBIC-L.

A3. For 3D staining with CUBIC-HV2, the FabuLight™ Fab fragment secondary antibody reacts with the primary antibody to create a complex before 3D staining. The two-step staining method (primary antibody → secondary antibody) commonly used for 2D sections is not recommended because it does not guarantee deep staining.

A4. We have verified the retention of the EGFP, EYFP, Venus, tdTomato, mCherry, mKate2 fluorescent signal. For other fluorescent proteins, please verify them yourself beforehand.

A1. CUBIC tissue clearing is designed to improve transparency and microscope resolution by slightly expanding the tissue. Thus, we recommend using tubes, containers, etc. with a diameter 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 used safely.

A2. Various cells may expand, but the physical relationship of the cells is maintained. Since the rigidness of the structure may vary depending on the tissue (nerves, veins, etc.) and this variation may cause artifacts, confirmation is required according to the situation.

A3.CUBIC tissue clearing reagents are optimized for PFA fixation. Lysis will occur on unfixed samples, so please fix the samples in advance.

A4. Samples that have been immersed in fixative for a long time (over several weeks) can still be cleared. However, since the fixation condition is a parameter that greatly affects the transparency of the samples, we recommend maintaining the preparation condition of the fixative and fixation period as much as possible. We recommend washing with PBS within 24 hours after perfusion fixation of the animal and promptly starting the CUBIC-L treatment.

A5. Paraffin-embedded samples can be cleared after deparaffinization treatment. Please refer to the following treatment method for more details.
Nojima, S., et al., Sci. Rep., 7(1), 9269(2017). [PMID:28839164]

A6. For whole-body clearing of a mouse, please prepare a sufficient volume of reagent to completely submerge the entire body. For organ clearing, please select a tube size that allow the entire organ to remain fully submerged when the tube is laid horizontally and filled with the reagent to approximately half its capacity. 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 the container used. As a rough estimate, 200 to 400 mL of CUBIC-L and 100 to 200 mL of CUBIC-R are required to clear the whole mouse body, and 20 to 40 mL of CUBIC-L and 10 to 20 mL of CUBIC-R is required to clear each mouse organ (approx. 1cm³).

A7. The following are possible causes. Please consider the following solutions.

1. High pH of the PFA solution used for fixation
   A pH exceeding 8.0 causes over-fixation, leading to yellowing of the organs and hindering the clearing process. Please ensure the pH is adjusted to between 7.0 and 7.5. Since clearing efficiency is affected by the pH and processing time of the PFA solution, it is recommended to standardize the fixation duration and preparation protocols.
2. Incomplete delipidation
   Consider extending the delipidation process or increasing the frequency of CUBIC-L exchange. We recommend exchanging CUBIC-L every two days and incubating at 37°C with shaking.
3. Incomplete clearing
   If moisture is carried over into CUBIC-R, the refractive index of the reagent will decrease, leading to insufficient clearing. Please replace the CUBIC-R with fresh reagent.

A8. For adult mouse tissues, the estimated delipidation period is approximately 3 to 7 days. However, the required time varies depending on the organ. Please determine the period based on the type and size of the organ, as well as your experimental objectives. For example, while light-sheet microscopy requires complete clearing, incomplete clearing in a shorter period may be sufficient for partial observations using two-photon microscope.

A1. Please consult with the waste management representative at your institution and follow the designated disposal procedures. As a general guideline, biological samples and reagents used for immersion should be disposed of as medical or infectious waste, while unused CUBIC reagents should be treated as organic liquid waste with high water content or flame-retardant liquid waste.

A2. Samples can be stored at room temperature while immersed in used CUBIC-R. However, to prevent potential solidification caused by the evaporation of water (the solvent in CUBIC-R), please ensure the container is tightly sealed with Parafilm or alternatives. Storage in agarose gel at room temperature is also possible and offers greater stability. For long-term archival storage, wash the samples with PBS after CUBIC-R treatment, and then store them at 4°C in PBS supplemented with a preservative such as sodium azide.
[Storage in Agarose Gel]
In an appropriate tube, add agarose powder to the CUBIC-R used for clearing to a final concentration of 2%, and dissolve by heating. Place the cleared sample into the solution before it cools and solidifies, then allow it to cool thoroughly until the agarose gel solidifies. For further details, please refer to the following paper.
Matsumoto, K., et al., Nat. Protoc., 14(12), 3506-3537(2019). [PMID:31748753]

A3. For the observation of cleared tissues, we recommend using light-sheet fluorescence microscope (LSFM), confocal laser scanning microscope (CLSM) equipped with objectives compatible with high refractive indices, or two-photon microscope. During observation, immerse the sample in a mounting medium for observation and use an objective lens that matches its refractive index. In recent years, affordable light-sheet microscopes specifically designed for observing cleared tissues have also been developed.
Reference: Otomo, K., et al., Nat. Commun., 15(1), 4941(2024). [PMID:38866781]

A4. The refractive index (RI) of CUBIC-R is 1.522. Avoid mixing CUBIC-R with water or other solvents, as this may alter the refractive index.

A5. CUBIC-1 (Sca/ eCUBIC-1, Reagent-1) and CUBIC-2 (Sca/ eCUBIC-2, Reagent-2) are different from CUBIC-L and CUBIC-R. CUBIC-1 and CUBIC-2 are the first generation of CUBIC reagents, while CUBIC-L and CUBIC-R are the improved second-generation reagents. The second generation offers significantly enhanced transparency. In terms of function, CUBIC-1 provides delipidation and decolorization like CUBIC-L, and CUBIC-2 serves as a refractive index (RI) adjusting agent, similar to CUBIC-R.