Feb 26, 2026
CUBIC-HistoVIsion2.0
- Etsuo A. Susaki1,
- Reo Kurusu1,
- Yuri Saito1
- 1Department of Biochemistry and Systems Biomedicine, Juntendo University Graduate School of Medicine
Protocol Citation: Etsuo A. Susaki, Reo Kurusu, Yuri Saito 2026. CUBIC-HistoVIsion2.0. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld1wyxl5b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 24, 2026
Last Modified: February 26, 2026
Protocol Integer ID: 240835
Keywords: organ immunostaining, termed cubic, cubic, histovision, available from cubicstar
Abstract
This protocol describes a method for simple and scalable whole-organ immunostaining, termed CUBIC-HistoVIsion 2.0 (CUBIC-HV2.0). The commercialized product developed based on this protocol are available from CUBICStars, Inc. (https://www.cubicstars.com/en/lineup/).
Guidelines
We strongly recommend that you first test this method with a benchmark experiment, specifically conducting anti-NeuN immunostaining with SYTOX-G nuclear staining on an entire mouse brain.
Materials
Reagents related to immunostaining by CUBIC-HV2.0
We recommend making 2× HV staining buffer to prepare 1× HV staining buffer containing HV-Additive.
- HV staining buffer: 10 mM HEPES [pH 7.5], 10% [w/v] Triton X-100, 500 mM NaCl, and 0.5% [w/v] casein
- HV wash buffer: 10 mM HEPES [pH 7.5], 10% [w/v] Triton X-100, 500 mM NaCl
- HV-LLPS reagent: Dimethyl silicone oil TSF451 (50 cSt, Momentive) or KF-96 (50 cSt, Shin-Etsu Silicon)
| A | B | C | |
| # in the screening | Name of the chemical | Vendor & Cat# | |
| CU#0146 | 2-[2-(Dimethylamino)ethoxy]ethanol | TCI, D1756 | |
| CU#0902 | 3-Methylamino-1,2-propanediol | TCI, M1243 | |
| CU#0854 | Nicotinic Acid Hydrazide | TCI, N0087 | |
| CU#0609 | N,N-Diethylnicotinamide | TCI, D0514 | |
| CU#0854 | Pyrazine | TCI, P0544 |
Reagents related to tissue clearing by CUBIC-L/R
- CUBIC-L: 10% [w/w] N-butyldiethanolamine (TCI, B0725) and 10% [w/w] Triton X-100 in distilled water
- CUBIC-R+: 45% [w/w] antipyrine (TCI, D1876), 30% [w/w] nicotinamide (TCI, N0078), 0.5% [v/v] N-butyldiethanolamine (TCI, B0725) in distilled water
Troubleshooting
Safety warnings
See safety data sheets for proper chemical handling, precautionary measures, and waste disposal.
Obey all local regulations/guidelines for handling and disposal of used reagents and solutions containing mixed reagents.
Ethics statement
Animal experiments should be approved by institutional committee and conducted according to institutional guidelines.
Before start
CUBIC-HV2.0 employs two technologies, HV-LLPS and HV-Additive, to enhance immunostaining. HV-LLPS concentrates the antibody-containing staining buffer into a skin-like layer surrounding the sample, increasing antibody concentration with a small amount of antibody (typically, 5–10 µg). HV-Additive modulates antigen-antibody interactions to improve antibody diffusivity inside the sample. HV-Additive is a cocktail of selected chemicals (typically, one or two chemicals), and optimal HV-Additive varies between antibodies. We list recommended antibodies that are validated by whole-organ staining and optimal experimental conditions for these antibodies.
| A | B | C | D | E | F | G | |
| Antibody | Tested tissue | 1st Ab | 2nd Fab | HV-Additive | Buffer Volume | Staining Period | |
| Synaptophysin (MBL, D073-3) | Brain | 10 µg | 4 µg | 2.5% #0146 | 100 µL | 13+1 days | |
| NeuN (Sigma, MAB377) | Brain | 5 µg | 4 µg | 2.5% #0854+5% #1086 | 100 µL | 5+1 days | |
| GAD (MBL, M018-3) | Brain | 3.5 µg | 4 µg | 2.5% #0854+5% #1086+1% #0146 | 70 µL | 11+1 days | |
| Th (Abcam, ab137869) | Brain | 3.5 µg | 4 µg | 2.5% #1086+5% #0609+1% #0146 | 70-100 µL | 7+1 days | |
| p-NF (BioLegend, 801601) | Brain | 10 µg | 4 µg | 1% #0902 | 50 µL | 10+1 days | |
| c-Fos (CST, 2250S) | Brain | 1 µg | 4 µg | 2.5% #0854 (+5% #1086) | 100 µL | 5+1 days | |
| c-Fos (CST, E2I7R) | Brain | 0.8–1 µg | 4 µg | 1.5% ＃854 | 100 µL | 6+1 days | |
| c-Fos (Abcam, ab208942) | Brain | 0.3 µg | 4 µg | 2.5wt% #0854 | 100 µL | 7+1 days | |
| c-Fos (Abcam, ab222699) | Brain | 0.5–1 µg | 4 µg | 1.5% ＃854 | 100 µL | 6+1 days | |
| c-Fos (Abcam, ab302685) | Brain | 0.5–1 µg | - | 1.5% ＃854 | 100 µL | 6+1 days @4˚C | |
| Gad67 (Sigma, MAB5406) | Brain | 3.5 µg | 4-6 µg | 2.5% #0854+5% #1086 | 70 µL | 6+1 days | |
| ChAT (Abcam, ab178850) | Brain | 2.5 µg | 4 µg | No Addtive | 100 µL | 6+1 days | |
| TPH2 (Sigma, AMAb91108) | Brain | 3.5 µg | 4 µg | 2.5% #0854+5% #1086 | 350 µL | 7+1 days | |
| Dat (Abcam, ab128848) | Brain | 2.5 µg | 4 µg | 2.5% #0854+5% #1086 | 350 µL | 6+1 days | |
| GFAP (MBL, D097-3) | Brain | 3.5 µg | 4-6 µg | 2.5% #1086+5% #0609 | 70 µL | 6+1 days | |
| GFAP (Sigma, G3893) | Brain | 5 µg | 4 µg | 1.5–2.5% #0854 | 100 µL | 7+1 days | |
| beta-amyloid (BioLegend, 93049) | Brain | 5 µg | 4-6 µg | 1.5% ＃0854 | 100 µL | 9+1 days | |
| beta-amyloid (IBL, 18584) | Brain | 5 µg | 4 µg | 2.5% #1086+5% #0609* | 50–70 µL | 15 +1days | |
| Phospho-Tau (Thermo, MN1020) | Brain | 5 µg | 4-6 µg | 2.5% 0854+5% 1086+1% 0146 | 60 µL | 11+1 days | |
| p-αSyn (Abcam, ab51253) | Brain | 5 µg | 4 µg | 2.5% ＃854 | 50 µL | 7+1 days | |
| Iba1 (WAKO, 019-19741) | Brain | 2.5 µg | 4 µg | 2.5% #0854 | >100 µL | 8+1 days | |
| PV (Swant, PV235) | Brain | 5 µL* | 4 µg | 2.5% #0854 | 50 µL | 7+1 days | |
| GFP (Abcam, ab290) | Brain** | 25 µg | 24 µg | 2.5% #0854 | 50 µL | 9+1 days | |
| GFP (Proteintech, gb2AF647) | Brain** | 0.5–1 µg | - | 2.5% #0854+5% #1086(+1% #0146) | 100 µL | 10 days | |
| α-SMA (Sigma, A5228) | Brain/Kidney | 5 µg | 4 µg | 2.5% #0854 | 50–100µL | 7+1 days | |
| CD31 (R&D, AF3628) | Brain/Tumor*** | 5 µg | 4 µg | 1.5% #0854 | 100 µL | 7+1 days | |
| AQP2 (Abcam, ab199975) | Kidney/Tumor*** | 5 µg | 2.8 µg | 2.5% #0854+5% #1086+1% #0146 | 50 µL | 14+1 days | |
| E-cadherin (BD, 610182) | Lung | 1 µg | 4 µg | No Addtive | 100 µL | 5+1 days | |
| VEGFR3 (R&D, AF743) | Lung | 1 µg | 4 µg | No Addtive | 100 µL | 5+1 days | |
| CD13 (Abcam, ab108310) | Tumor*** | 3 µg | 3 µg | 2.5% #0854 + 5% #1086 | 30 µL | 6+0 days | |
| CD13 (R&D, AF2335) | Tumor*** | 3 µg | 0.75 µg | 2.5% #0854 + 5% #1086 | 30 µL | 4–5 days | |
| Vimentin (CST, 5741) | Tumor*** | 1 µg | 1 µg | 2.5% #0854 + 5% #1086 | 30 µL | 4+1 days | |
| HuNu (Sigma, MAB1281) | Tumor*** | 1 µg | 1 µg | 1.5% ＃0854 | 30 µL | 4+1 days |
* Ab concentration was not determined for this Ab.
** Anti-GFP Ab was validated using GAD67-GFP (for Proteintech, gb2AF647) or Thy1-GFP-M (for Abcam, ab290) mouse brain.
*** 2 mm-thick tissues of Cell line-derived xenograft (LS174T or BT474) were used.
Perfusion fixation, dissection of the mouse organ, and post-fixation
17h 30m
Anesthetize the mouse.
5m
Transcardially perfuse 10 mL (4 mL/min) of ice-cold PBS containing 10 U/mL of Heparin.
5m
Transcardially perfuse 20 mL (6 mL/min) of ice-cold 4% (w/v) PFA.
5m
Dissect the organs.
15m
Post-fix the dissected organs in 4% (w/v) PFA in PBS (~10 mL /whole organ) for Overnight at 4 °C with gentle shaking (40 rpm to 50 rpm ).
16h
Wash the sample in PBS containing 0.05% NaN3 for 01:00:00 × 3 times at Room temperature with gentle shaking (40 rpm to 50 rpm ).
1h
Delipidation
4d 22h
Immerse the fixed sample in 10 mL of 0.5× CUBIC-L (1:1 dilution with water) in the 30 mL tube and incubate it Overnight at 37 °C with gentle shaking (40 rpm to 50 rpm ).
16h
Replace with 10 mL to 15 mL of 1× CUBIC-L in the 30 mL tube and delipidate for 72:00:00 to 120:00:00 (3 to 5 days) at 37 °C with gentle shaking (40 rpm to 50 rpm ).
Note
Replace CUBIC-L every 48:00:00 to 72:00:00 (2 to 3 days) if the duration of treatment exceeds 72:00:00 (3 days).
4d
Wash the sample with 20 mL of PBS containing 0.05% NaN3 for 02:00:00 × 3 times (or 02:00:00 ×1, Overnight ×1, 02:00:00 ×1) at 37 °C with gentle shaking (40 rpm to 50 rpm ).
Note
- After each use, the tubes should be washed or replaced in order to remove Triton X-100 thoroughly.
- The delipidated sample can be stored in PBS containing 0.05% NaN3 at 4 °C for at least several months.
6h
3D staining with HV-LLPS and HV-Additive
1w 1d 23h 42m
Immerse the delipidated sample in 10 mL of PBS containing 5% MeOH in the 15 mL standing tube and incubate it for Overnight at 37 °C with gentle shaking (40 rpm to 50 rpm ).
16h
Exchange the buffer to 10 mL of HV staining buffer (10 mM HEPES [pH 7.5], 10% [w/v] Triton X-100, 500 mM NaCl, and 0.5% [w/v] casein) containing HV-Additive suitable for your primary Ab and incubate it for 01:30:00 at 37 °C with gentle shaking (40 rpm to 50 rpm ).
1h 30m
During the step 11, prepare primary Ab + secondary Fab fragment mix as follows:
- Calculate the required volume (X, Y) of primary Ab and secondary Fab fragment. Example of calculation is shown below.
| A | B | C | D | |
| Primary or Secondary | Required Amount | Product Concentration | X or Y Volume | |
| Primary Ab | 5 µg | 1 mg/mL | 5 µL | |
| Secondary Fab | 4 µg | 1.5 mg/mL | 2.7 µL |
- Prepare the following 3D staining solution (typically 50 to 100 µL per whole mouse brain). Example of preparation is shown below.
| A | B | |
| Reagent | Volume | |
| 2× HV staining buffer containing HV-Additive | 50 µL | |
| Primary Ab | X µL | |
| Secondary Fab | Y µL | |
| Nuclear stain* | Z µL | |
| Distilled water | 50-(X+Y+Z) µL | |
| Total | 100 µL |
Note
- The total volume of the staining solution should be determined based on the quantity and final concentration of primary Ab. For instance, in the case of using 5 µg of primary Ab at a concentration of 100 µg/mL, a volume of 50 µL should be prepared for the 3D staining solution (5 µg of primary Ab in 50 µL = 100 µg/mL). Generally, 50-100 µg/mL of antibody concentration prepared in 50-350 µL of the staining solution is recommended.
- The type of HV-additives should be determined for each primary antibody (please check "Before start" section). Generally, one or two HV-Additive reagent are used.
Note
*Recommended amount of nuclear stain
BOBO-1 Iodide: 4 µL/whole mouse brain
SYTOX Green Nucleic Acid Stain: 1.6 µL/whole mouse brain
RedDot2 Far-Red Nuclear Stain: 4 µL/whole mouse brain
- Add the entire volume to the 500 µL Protein LoBind tube and incubate the mix for 01:30:00 at 37 °C .
5m
Note
This 3D tissue staining pot for CUBIC-HV2.0 can be used repeatedly. After the usage, wash the pot with neutral detergent and distilled water.
1m
Collect the sample with a metal spoon and put it into the staining pot.
Note
A portion of the sample should be attached gently on soft paper towel to absorb residual buffer.
1m
Slowly pour the HV-LLPS reagent into the staining pot until the pot is filled. Remove the bubbles.
Note
HV-LLPS prevents samples from drying out during staining by covering the entire sample and the staining solution, which contains condensed staining probes.
1m
Close the lid tightly and incubate the chamber for at least 168:00:00 (1 week) at Room temperature with slow rotation (<1 rpm ) under a light-shielded condition.
1w
To stabilize the 2nd Fab signals, further incubate the staining chamber for 24:00:00 at 4 °C with slow rotation (<1 rpm ) under a light-shielded condition.
1d
One hour before moving to the step 19, prepare HV wash buffer (10 mM HEPES [pH 7.5], 10% [w/v] Triton X-100, 500 mM NaCl) and cool it On ice .
1m
Recover the sample from the staining pot with a metal spoon. Discard the staining solution and the HV-LLPS reagent.
1m
Remove the residual buffer and the HV-LLPS reagent as in step 14.
1m
Immerse the sample in 10 mL of pre-cooled HV wash buffer in the 15 mL standing tube. Gently invert the tube several times to separate the wrapping reagent remaining on the sample surface.
1m
Replace the HV wash buffer with a new one and continue washing the sample gently at 4 °C with light shielding, while shaking at 40 rpm to 50 rpm , for 02:00:00 .
2h
Replace the HV wash buffer with the new one and continue washing the sample two more times (02:00:00 ×2).
4h
Post-fixation with 1% FA
1d 3h 5m
During the sample wash, prepare up to 10 mL of fixative solution by diluting formalin (FA) solution to 1% in HV wash buffer and cool it On ice .
Note
The saturated formalin solution contains 35 to 38% of formalin. For example, when using a 37% formalin solution, dilute it with HV wash buffer at a ratio of 1:36.
5m
Fix the sample in the 1% FA for 24:00:00 at 4 °C with gentle shaking (40 rpm to 50 rpm ) under a light-shielded condition.
1d
To accelerate the fixation reaction, further incubate the sample for 01:00:00 at 37 °C with gentle shaking (40 rpm to 50 rpm ) under a light-shielded condition.
Note
Reducing the FA reaction time in steps 25 and 26 could result in the attenuation of antibody signals.
1h
Wash the sample in 30 mL of PBS in the 50 mL tube for 02:00:00 at Room temperature with gentle shaking (40 rpm to 50 rpm ) under a light-shielded condition.
2h
RI matching with CUBIC-R+
3d
Incubate the sample in 15 mL of 0.5× CUBIC-R+ (1:1 diluted with distilled water) in a 30 mL tube for 24:00:00 at >25 °C with gentle shaking (40 rpm to 50 rpm ) under a light-shielded condition.
1d
Remove the residual diluted CUBIC-R+ as in section 3-step 5 and incubate the sample in 15 mL (or 30 mL if the tissue will be embedded in agarose gel) of 1× CUBIC-R+ for 48:00:00 (2 days) at >25 °C with gentle shaking (40 rpm to 50 rpm ) under a light-shielded condition.
Note
- Carryover of residual diluted CUBIC-R+ results in a poor clearing result due to a decrease in the final refractive index.
- CUBIC-R+ crystallizes easily in winter. Ensure that sample in the reagent are handled and incubated at 25°C or higher.
- If required, CUBIC-R+-cleared samples can be embedded in 2% agarose/CUBIC-R+. For more details, please refer to our protocol paper and our resource website.
2d
Use the sample for microscopic observation (gel embedding if necessary)