CUBIC-HV

Whole body scaled 3D staining reagent kit
CUBIC-HV is a revolutionary new technology that enables a 3D tissue staining and observed in all organs and systemic scale. (RIKEN / University of Tokyo Professor Ueda and Susaki Associate Professor development) This protocol is intended to provide a highly efficient 3-dimensional tissue staining technique. Co., Ltd. CUBICStars is licensed on CUBIC-HV, it has been manufacturing the reagent kit. According to the application offers the following 2 products

＜Note＞
Differences between CUBIC-HV™1 3D Immunostaining kit (product code: C3708) and CUBIC-HV™1 3D immunostaining kit (casein separately) (product code: C3717) ;
The CUBIC-HV™1 3D Immunostaining kit (product code: C3708) contains casein dissolved in immunostaining buffer and can be used immediately. The immunostaining buffer should be used as soon as possible.
CUBIC-HV™1 3D immunostaining kit (Casein separately) (product code: C3717) does not contain casein dissolved in the immunostaining buffer and is enclosed in a separate package. Mix casein into the immunostaining buffer immediately before use, and use the buffer as soon as possible after adding casein. It takes about 2 hours at room temperature to dissolve the casein.

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Product code	Product name	Package	SDS / Protocol
C3709	CUBIC-HV™1 3D nuclear staining kit	1 kit	SDS Protocol
C3708	CUBIC-HV™1 3D immunostaining kit	1 kit	SDS Protocol
C3717	CUBIC-HV™1 3D immunostaining kit (Casein separately)	1 kit	SDS Protocol

Product information

Whole body scaled 3D staining reagent kit

CUBIC-HV™1 3D Immunostaining kit (product code: C3708)

Process outline

Delipidation

Perfusion fixation and dissection of the mouse brain
Post-fix <24 h
Wash (PBS) 2 h x3
Delipidation (CUBIC-L; Product No. T3740) 3-7 days
Wash (PBS) 2 h x3

Nuclear staining by CUBIC-HV™1 3D nuclear staining kit (Product No. C3709)

Nuclear staining (DAPI or Propidium Iodide or others in CUBIC-HV™1 3D nuclear staining buffer) 3-5 days
Wash (3D nuclear staining wash buffer; 10mM HEPES buffer, pH 7.5) 2 h x3

Optional process; Enzyme reaction

Immunostaining by CUBIC-HV™1 3D immunostaining kit (Product No. C3708)

Primary antibody (anti-NeuN antibody in C3708 kit can be used): 5 µg/whole brain
Secondary antibody: anti-Fc Fab (out of the kits), 3.75 µg/whole brain
Primary antibody + secondary antibody reaction / Replacement with CUBIC-HV™1 3D immunostaining buffer, 1.5 h
Immunostaining (CUBIC-HV™1 3D immunostaining buffer with CUBIC-HV™1 3D immunostaining additive (if required)), >1 week
Reaction in the same buffer at 4°C, 1 day
Wash (CUBIC-HV™1 3D immunostaining wash buffer), 2 h x3

Fix and RI matching

Post-fix (in 1% Formaldehyde Solution), 1 day
Wash (PBS) 2 h
RI matching (CUBIC-R+; Product No. T3740), <3 days
Microscopic observation

References

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.

Product information

Chemical Reagents
(Lithium Battery Grade)

Chemical Reagents/
Test-kits
(Life-Science Industry)

High-purity Chemicals/
General reagents