CUBIC

• Easy-to-use
• No special equipment required
• Follow up 2D H&E
• High transparency
• Applicable for many organs
• Compatible with IF, FP and other fluorescent labels

Whole organ samples

2 mm section samples

Fluorescent protein expression tissue

Whole organ samples

**Typically, 3:7 mixture of Mounting Solution 1 and 2 (RI ~1.49) can be used.

	Brain	Heart	Lung	Kindney	Liver
PBS
CUBIC

Figure1.Transmittance images of mouse whole organs before and after clearing with Scale CUBIC Solutions.

2 mm section samples

**Typically, 3:7 or 2:8 mixture of Mounting Solution 1 and 2 (RI ~1.49) can be used

	Brain	Heart	Liver
PBS
CUBIC

Figure2.Transmittance images of mouse 2mm slices before and after clearing with Scale CUBIC Solutions.

Fluorescent protein expression tissue

Figure3. LSFM imaging of CAG-EGFP Tg Mouse (8w Male) whole brain, whole heart and dissected liver.

CUBIC Application with pathological methods

CUBIC Application with fish clearing

CUBIC Application with crustacean clearing

CUBIC Application with beetle clearing

Visualization of whole-brain vascular networks

CUBIC Application with pathological methods

Data by S. Nojima.et al. : Scientific Reports ,7,9269 (2017) / Adapted.

	Before CUBIC		After CUBIC -------------------------------------------------------------------------------------------
		0 day	2 days	3 days	4 days	6 days	7 days
Human lung	CUBIC
	HE stain
Human Lymph node	CUBIC
	HE stain

Figure 4.Tissue clearing of various human organs with CUBIC
Tissue clearing and 3D imaging of deparaffinizedsamples with CUBIC

Figure 5.Tissue clearing and 3D imaging of pathological specimens with CUBIC.

CUBIC Application with fish clearing

Data were provided by Kunihiko Futami, Tokyo University of Marine Science and Technology.

Figure6. Fish clearing with CUBIC
See Reference 5) for the protocol

Figure7. Stained image of Fish organ treated by CUBIC
Organs were sliced at 5 μm and stained with Mayer's hematoxylin after CUBIC treatment.

Figure8. Immunostaining image of zebrafish treated with CUBIC

CUBIC Application with crustacean clearing

Data were provided by Aru Konno and Shigetoshi Okazaki, Hamamatsu University School of Medicine in Japan.

Figure9. Crustacean clearing with CUBIC
See Reference 6) for the protocol

Figure10. Crustacean clearing with CUBIC (PI staining)
See Reference 6) for the protocol

CUBIC Application with beetle clearing

Data were provided by Shinya Kuroda, Tokyo University

Beetle clearing requires fixing, bleaching, and clearing steps.
See the website for Detailed protocols.

Figure11. Beetle clearing with CUBIC
LEFT: drone beetle MIDDLE: rhinoceros beetle RIGHT: stag beetle
This protocol altered from reference 7).

Visualization of whole-brain vascular networks

Data by Miyawaki, T. et al. : Nat. Commun., 11：(1)　(2020)

Figure12. Tissue clearing with sodium deoxycholate (SDC)
TOP: SDS-based delipidation. The tissue was swelling.
BOTTOM: SDC-based delipidation. Tissue swelling was suppressed.
This protocol altered from reference 8).

Figure13. Mouse brain cortex clearing after vascular casting
LEFT: SDS-based delipidation. Some penetrating vessels were severed (arrowheads).
BOTTOM: SDC-based delipidation. Breakages of vessels were rarely.
This protocol altered from reference 8).

1. E. A. Susaki. et al. : Cell, 157(3), 726 (2014).
2. K. Tainaka. et al. : Cell, 159(4), 911 (2014).
3. E. A. Susaki. et al. : Nature Protocols, 10, 1709 (2015).
4. S. Nojima. et al. : Scientific Reports, 7, 9269 (2017).
5. S. Ohnuma. et al.: Fish Pathology, 52(2), 96(2017).
6. A. Konno and S. Okazaki.: Zoological Letters, 4:13(2018).
7. M Kuroda and S Kuroda (2020). Whole-body clearing of beetles by successive treatment of hydrogen peroxide and CUBIC reagents.
8. Miyawaki, T. et al. : Nat Commun., 27, 11(2020).