CUBIC-HV™1 3D immunostaining kit (Casein separately)

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This kit contains buffers and anti-NeuN antibody (positive control) for 3D nuclear staining of CUBIC-cleared tissues.

The difference between CUBIC-HV™1 3D immunostaining kit (Product No. C3708) and CUBIC-HV™1 3D immunostaining kit (Casein separately) (Product No. C3717):
CUBIC-HV™1 3D immunostaining kit (Product No. C3708) includes a buffer with casein, which can be used as soon as it is arrived. The buffer with casein should be used as soon as possible.
CUBIC-HV™1 3D immunostaining kit (Casein separately) (Product No. C3717) includes a buffer without casein and casein is in the kit separately. The casein should be mixed to the buffer just before use and after mixing, the buffer should be used as soon as possible. It takes about 2 hours to solve the casein.

Please see the detail protocols in CUBICStars’ web site.
https://www.cubicstars.com/cubic-hv/index.html

Constituents in kit (suitable for a brain of an adult mouse)

• CUBIC-HV™1 3D immunostaining buffer (without Casein) (2x) (for 10 tests)
• CUBIC-HV™1 3D immunostaining wash buffer (1x) (for 10 tests)
• CUBIC-HV™1 3D immunostaining additive (10x) (for 10 tests)
• Anti-NeuN Mouse IgG1 antibody (1mg/mL) (for 2 tests)
• 10x 15 mL tube

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 (Casein separately) (Product No. C3717) ====

Add the entire amount of casein powder in the kit (1 vial) to the 3D immunostaining buffer (without Casein). It takes about 2 hours at room temperature.
From this time on, 3D immunostaining buffer including Casein should be used.

Primary antibody (anti NeuN antibody in C3717 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+(M); Product No. T3740), <3 days
Microscopic observation

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