CUBIC-HVTM2 staining protocol
for whole mouse organs(version 2024-03)

CUBICStars, Inc.
All rights reserved.

Product outline
Histological staining of three-dimensional volumetric tissue samples can be highly challenging. This is due tocomplications associated with the deep penetration of staining agents and antibodies within a volumetrictissue sample. CUBIC-HistoVIsion2 has enabled versatile three-dimensional staining by resolving theseissues. Important factors for 3D staining were identified in the research: 1) the concentration of the staining
probe in the buffer, and 2) the appropriate modulation of probe-tissue interactions.
Based on the most recent version of the CUBIC-HistoVIsion protocol1, our patented product, CUBIC-HVTM2,
integrates two innovative technologies to address these critical factors:
HV-LLPS★ is an innovative technique that increases the staining probe concentration through the reductionof the staining buffer volume to a minimum degree. By significantly condensing the staining probes (50-100µg/mL for each primary antibody), this technology permits the deep staining of three-dimensional tissues inan efficient manner.
HV-additive★ is a set of ideal compounds that significantly enhance staining probe penetration, screened
from a library of more than 500 compounds. By modulating probe-tissue interactions, deep diffusion ofstaining probes into three-dimensional tissues is strongly supported.

CUBIC-HVTM2 enables your three-dimensional staining experiments for a variety of purposes without
requiring specialized equipment or reagents that are not readily accessible.

NOTE
Generally, performing 3D staining on large tissue samples poses a challenge,
even for users well-versed in histological techniques. We strongly advise
initiating the application of this method with a benchmark experiment,
specifically conducting anti-NeuN immunostaining with SYTOX-G nuclear
staining on an entire mouse brain. Should you encounter any difficulties, please
do not hesitate to reach out to our expert team for assistance. We also welcome
any feedback from customers to improve this instruction.

Inquiry to our experts

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Kit components
1× CUBIC-HV™2 Pre-wash buffer (150 mL)
2× CUBIC-HV™2 Staining buffer (80 mL)
10× CUBIC-HV™2 Additive A (16 mL)
10× CUBIC-HV™2 Additive B (16 mL)
10× CUBIC-HV™2 Additive C (16 mL)
10× CUBIC-HV™2 Additive D (16 mL)
10× CUBIC-HV™2 Additive E (16 mL)
10× CUBIC-HV™2 Additive F (16 mL)
CUBIC-HV™2 Wrapping reagent (35 mL)
2× CUBIC-HV™2 Wash buffer (300 mL)
Casein Sodium (0.8 g)**The day prior to usage, dissolve casein in 2× CUBIC-HV™2 Staining buffer through robust stirring using astirrer.
■ No heating is necessary.
■ Avoid adding all the casein simultaneously; instead, introduce it gradually in multiple portions.
Adding it all at once may lead to casein aggregation, resulting in incomplete dissolution.
■ Do not employ a vortex, as the buffer contains a high concentration of detergent, which could leadto foaming.

Additional reagents to be used
PBS (Tablet): TaKaRa #T9181
Sodium azide (NaN3): Nacalai tesque #31208-82
Paraformaldehyde (PFA): Nacalai tesque #02890-45
Formalin solution: Nacalai tesque #16222-65
Heparin: FUJIFILM Wako #081-00136
Tissue-Clearing Reagent CUBIC-L: CUBICStars #CSCR001
Tissue-Clearing Reagent CUBIC-R+(N): CUBICStars #CSCR002

Recommended nuclear stains
Choose a suitable nuclear stain for your experimental design:・Blue-green: BOBO™-1 Iodide (462/481), ThermoFisher Scientific #B3582
Can be used with yellow-orange (e.g., AF532, Cy3, tdTomato), orange-red (e.g., AF594, mCherry), andred-far red (e.g., AF647) dyes and fluorescent proteins
・Green: SYTOX™ Green Nucleic Acid Stain, ThermoFisher Scientific #S7020
Can be used with orange-red (e.g., AF594, mCherry), and red-far red (e.g., AF647) dyes and fluorescentproteins
・Red-far red: RedDot™2 Far-Red Nuclear Stain, biotium #40061
Can be used with green (e.g., FITC, GFP, YFP), yellow-orange (e.g., AF532, Cy3, tdTomato), andorange-red (e.g., AF594, mCherry) dyes and fluorescent proteins

Antibodies
Primary antibody■ It is recommended to use antibody products containing a concentration of 250 µg/mL or higher.
Secondary antibody
FabuLightTM: Fc specific Fab fragment (Jackson Immunolab)
https://www.jacksonimmuno.com/catalog/31#target:15■ Alexa Fluor® 488 is quenched in CUBIC-R. We recommend FabuLightTM products labeled with Cy3,
Alexa Fluor® 594, or Alexa Fluor® 647.
■ Alternatively, direct dye-conjugated primary antibodies may be employed. The secondary
antibody is unnecessary in this case.

Containers to be used
Protein LoBind 500 µL tube: Eppendorf #022431064
5 mL tube: Eppendorf #0030119401
15 mL standing tube: SARSTEDT #60.732.001
30 mL tube: SARSTEDT #60.544
50 mL tube: Falcon #352070
3D tissue staining pot for CUBIC-HV™2: CUBICStars #CSSR005

Process outline
Perfusion fixation and dissection of the mouse organ↓
Post-fix↓ 18~24 h at 4°C
Wash (PBS)↓ 2 h ×3 at RT
Delipidation (CUBIC-L)↓ 3~7 days at 37°C
Wash (PBS/NaN3)↓ 2 h ×3 at 37°C → (Can be stored at 4°C)
Pre-wash (CUBIC-HV™2 Pre-wash buffer)↓ overnight at 37°C
Primary antibody + secondary Fab reaction
Exchange to the CUBIC-HV™2 Staining buffer containing HV-additives★
↓ 1.5 h at 37°C
Nuclear & immunostaining (CUBIC-HV™2 Staining buffer with HV-LLPS★ & HV-additives★)↓ 1 week~ at RT
Reaction at 4°C↓ 1 day
Wash (CUBIC-HV™2 Wash buffer)↓ 1.5 h ×3 at 4°C
Post-fix (1% FA in CUBIC-HV™2 Wash buffer)↓ 24 h at 4°C → 1 h at 37°C
Wash (PBS)↓ 2 h at RT
RI matching (CUBIC-R+)↓ 50% diluted CUBIC-R+ overnight at >25°C↓ 100% CUBIC-R+ 2 days~ at >25°C(Gel embedding)↓
Microscopic observation

Process details* The reaction time and quantity of reagent required to stain a single whole mouse brain are detailed
in this protocol.
■ With the exception of staining and the 4°C reaction, every shaking phase is executed in thehorizontal orientation.
■ Delipidated organ samples are susceptible to injury and should be handled with a metal spoon.
1) Collection of mice organs3,4
1. Anesthetize the mouse.
2. Transcardially perfuse 10 mL (4 mL/min) of ice-cold PBS containing 10 U/mL of Heparin.
3. Transcardially perfuse 20 mL (6 mL/min) of ice-cold 4% (w/v) PFA.
4. Dissect the organs.
5. Post-fix the dissected organs in 4% (w/v) PFA in PBS (~ 10 mL/whole organ) for overnight (8-24 h) at
4°C with gentle shaking (40-50 rpm/min).
6. Wash the sample in PBS (+0.05% NaN3) for 3 h × 3 times at room temperature with gentle shaking (4050 rpm/min).
2) Delipidation with CUBIC-L3
1. Immerse the fixed sample in 10 mL of 0.5× CUBIC-L (1:1 dilution with water) in the 30 mL tube andincubate it for overnight at 37°C with gentle shaking (40-50 rpm/min).
2. Replace to 10-15 mL of 1× CUBIC-L in the 30 mL tube and delipidate for 3-5 days at 37°C with gentle
shaking (40-50 rpm/min).
■ Replace CUBIC-L every 2 to 3 days if the duration of treatment exceeds 3 days.
3. Wash the sample with 20 mL of PBS containing 0.05% NaN3 for 2 h × 3 times (or 2 h ×1, overnight ×1, 2h ×1) at 37°C with gentle shaking (40-50 rpm/min).
■ 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/0.05% NaN3 at 4°C at least for several months.
3) 3D staining with HV-LLPS★ and HV-additives★
1. Immerse the delipidated sample in 10 mL of 1× CUBIC-HV™2 Pre-wash buffer in the 15 mL standing
tube and incubate it for overnight at 37°C with gentle shaking (40-50 rpm/min).
2. Exchange the buffer to 10 mL of 1× CUBIC-HV™2 Staining buffer containing selected 1× HV-additive★
reagents and incubate it for 1.5 h~ at 37°C with gentle shaking (40-50 rpm/min).
■ The type of HV-additives★ should be determined for each 1st antibody. Generally, one HVadditives★ reagent or two are used.
For 10 mL of the 1× CUBIC-HV™2 Staining buffer containing a single HV-additive★ reagent:
2× CUBIC-HV™2 Staining buffer
5 mL (final 1×)
10× HV-additives★ reagent
1 mL (final 1×)
Distilled water
4 mL============================================================
Total
10 mL
3. During the step 2, prepare 1st Ab + 2nd Fab fragment mix as follows.
a. Calculate the required volume (X, Y) of 1st Ab and 2nd Fab fragment.
1st Ab: 1-5 µg/whole mouse brain (approximately 0.6 g)
In the case of using 5 µg of 1 mg/mL (= 1 µg/µL) 1st Ab product, X = 5 (µg) /1 (µg/µL) = 5 µL.
2nd Fab fragment: 4 µg/whole mouse brain
In the case of using 1.5 mg/mL product, Y = 4 (µg)/1.5 (µg/µL) ≒ 2.67 µL.
■ When using a direct dye-conjugated primary antibody, 3-a can be skipped.
■ The amount and the final concentration of 1st Ab should be determined for each application.
■ When applying to organs other than the brain, estimate the required quantity of antibodies andreagents by considering the weight ratio of the organ.

b. Prepare the following 3D staining solution (typically 50-100 µL per whole mouse brain).
■ The total volume of the staining solution should be determined based on the quantity and final
concentration of 1st Ab. For instance, in the case of employing 5 µg of 1st Ab at a concentrationof 100 µg/mL, a volume of 50 µL should be prepared for the 3D staining solution (5 µg of 1st 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 1st antibody (the guide is provided
on our website). Generally, one HV-additives★ reagent or two are used.
For 100 µL of the 3D staining solution containing a single HV-additive★ reagent:
2× CUBIC-HV™2 Staining buffer
50 µL (final 1x)
1st AbX µL
2nd Fab fragment
Y µL
Nuclear stain*
Z µL
10× HV-additives★ reagent
10 µL
Distilled water
50 - (X+Y+Z+10) µL============================================================
Total
100 µL
Add the entire volume to the 500 µL Protein LoBind tube and incubate the mix for 1.5 h at 37°C.
*Recommended amount of nuclear stain
BOBO™-1 Iodide (462/481): 4 µL/whole mouse brain
SYTOX™ Green Nucleic Acid Stain: 1.6 µL/whole mouse brain
RedDot™2 Far-Red Nuclear Stain: 4 µL/whole mouse brain
4. After the reaction in step 3, transfer the entire 3D staining solution into the 3D tissue staining pot[CSSR005].
■ This 3D tissue staining pot for CUBIC-HV™2 can be used repeatedly. After the usage, wash the
pot with a neutral detergent and distilled water.
5. Collect the sample with a metal spoon and put it into the staining pot.
■ A portion of the sample should be attached gently on soft paper towel to absorb residual buffer.

6. Slowly pour CUBIC-HV™2 Wrapping reagent into the staining pot until the pot is filled. Remove thebubbles.
■ The wrapping reagent prevents samples from drying out during staining by covering the entire
sample and the staining solution containing condensed staining probes (HV-LLPS★).

7. Close the lid tightly and incubate the chamber for 1 week~ at RT with slow
rotation (<1 rpm) under a light-shielded condition.
■ The staining period should be determined for each antibody.

8. To stabilize the 2nd Fab signals, further incubate the staining chamber for 24 h at 4°C with slow rotation(<1 rpm) under a light-shielded condition.
9. 1 h before moving to the step 10, prepare 1× CUBIC-HV™2 Wash buffer and cool it on ice.
For 40 (10 × 4) mL of the 1× CUBIC-HV™2 Wash buffer:
2× CUBIC-HV™2 Wash buffer
20 mL (final 1×)
Distilled water
20 mL============================================================
Total
20 mL
10. Recover the sample from the staining pot with a metal spoon. Discard the staining solution and thewrapping reagent.
11. Remove the residual buffer and the wrapping reagent as in step 5.
12. Immerse the sample in 10 mL of pre-cooled 1× CUBIC-HV™2 Wash buffer in the 15 mL standing tube.
Gently invert the tube several times to separate the wrapping reagent remaining on the sample surface.
13.Replace the 1× CUBIC-HV™2 wash buffer with a new one and continue washing the sample gently at
4°C with light shielding, while shaking at 40-50 rpm/min, for 2 h.
14. Replace the wash buffer with the new one and continue washing the sample two more times (2 h ×2).
4) Post-fix with 1% FA
1. During the sample wash, prepare 7~8 mL of fixative solution by diluting formalin (FA) solution to final 1%
in 1× CUBIC-HV™2 Wash buffer and cool it on ice.
■ The saturated formalin solution contains 35 to 38% of formalin. For example, when using a 37%
formalin solution, dilute it with 1x CUBIC-HV™2 Wash buffer at a ratio of 1:36.
For 7.4 mL of the FA solution:
2× CUBIC-HV™2 Wash buffer
3.7 mL (final 1×)
Saturated formalin (37%)*
0.2 mL (final 1%)*The concentration may be different by products.
Distilled water
3.5 mL============================================================
Total
7.4 mL
2. Fix the sample in the 1% FA for 24 h at 4°C with gentle shaking (40-50 rpm/min) under a light-shielded
condition.
3. To accelerate the fixation reaction, further incubate the sample for 1 h at 37°C with gentle shaking (4050 rpm/min) under a light-shielded condition.
■ Reducing the FA reaction time in steps 2 and 3 could result in the attenuation of antibody signals.
4. Wash the sample in 30 mL of PBS in the 50 mL tube for 2 h at RT with gentle shaking (40-50 rpm/min)under a light-shielded condition.

5) RI matching with CUBIC-R+
1. Incubate the sample in 15 mL of 0.5× CUBIC-R+ (1:1 diluted with distilled water) in a 30 mL tube for 24
h at >25°C with gentle shaking (40-50 rpm/min) under a light-shielded condition.
2. Remove the residual diluted CUBIC-R+ as in section 3-step 5 and incubate the sample in 15 mL (or 30
mL if gel embedding is applied) of 1× CUBIC-R+ for 2 days at >25°C with gentle shaking (40-50 rpm/min)under a light-shielded condition.
■ 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 andincubated 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 the original paper (ref. 3) and RIKEN CUBIC resource website (http://cubic.riken.jp/)
3. Use the sample for microscopic observation (gel embedding if necessary)

CUBIC-HVTM2Guide for evaluating primary antibodies & HV-additive★
Reagents to be used
PBS (Tablet): TaKaRa #T9181
Sodium azide (NaN3): Nacalai tesque #31208-82
Paraformaldehyde (PFA): Nacalai tesque #02890-45
Formalin solution: Nacalai tesque #16222-65
Heparin: FUJIFILM Wako #081-00136
Sucrose: Nacalai tesque #30403-55
DAPI (1 mg/mL solution): Thermo Fisher #62248
Tissue-Clearing Reagent CUBIC-L: CUBICStars #CSCR001
2× CUBIC-HV™2 Staining buffer
10× CUBIC-HV™2 Additive A
10× CUBIC-HV™2 Additive B
10× CUBIC-HV™2 Additive C
10× CUBIC-HV™2 Additive D
10× CUBIC-HV™2 Additive E
10× CUBIC-HV™2 Additive F
2× CUBIC-HV™2 Wash buffer
Casein Sodium**The day prior to usage, dissolve casein in 2× CUBIC-HV™2 Staining buffer through robust stirring using astirrer, according to the CUBIC-HV™2 general protocol.
Cryopreservation reagent
40(w/v)% Sucrose (100 mL)：dissolve 40 g of sucrose in PBS and adjust the total volume to 100 mL.
25(w/v)% Sucrose (100 mL)：dissolve 25 g of sucrose in PBS and adjust the total volume to 100 mL.

Antibodies
Primary antibody■ It is recommended to use antibody products containing a concentration of 250 µg/mL or higher.
Secondary antibody
FabuLightTM: Fc specific Fab fragment (Jackson Immunolab)
https://www.jacksonimmuno.com/catalog/31#target:15■ Alexa Fluor® 488 is quenched in CUBIC-R. We recommend FabuLightTM products labeled with Cy3,
Alexa Fluor® 594, or Alexa Fluor® 647.
■ Alternatively, direct dye-conjugated primary antibodies may be employed. The secondary
antibody is unnecessary in this case.

Containers to be used
Protein LoBind 500 µL tube: Eppendorf #022431064
15 mL standing tube: SARSTEDT #60.732.001
3D tissue staining pot for CUBIC-HV™2: CUBICStars #CSSR005
24 well plate：Falcon #353047

Process outline
Perfusion fixation and dissection of the mouse organ↓
Post-fix↓ 18~24 h at 4°C
Wash (PBS)↓ 2 h ×3 at RT
Delipidation (CUBIC-L)↓ 3~7 days at 37°C
Wash (PBS/NaN3)↓ 2 h ×3 at 37°C → (Can be stored at 4°C)
Prepare cryosections↓ wash → (Can be stored at 4°C)
STEP 1: Evaluate primary antibodies (2D staining)
STEP 2: Evaluate suitable HV-additives★ for each primary antibody (2D staining)
STEP 3: Determine 3D staining condition for each primary antibody↓
Prepare cryosections

Process details< Sample preparation >1) Collection of mice organs3,4
1. Anesthetize the mouse.
2. Transcardially perfuse 10 mL (4 mL/min) of ice-cold PBS containing 10 U/mL of Heparin.
3. Transcardially perfuse 20 mL (6 mL/min) of ice-cold 4% (w/v) PFA.
4. Dissect the organs.
5. Post-fix the dissected organs in 4% (w/v) PFA in PBS (~ 10 mL/whole organ) for overnight (8-24 h) at
4°C with gentle shaking (40-50 rpm/min).
6. Wash the sample in PBS (+0.05% NaN3) for 2 h × 3 times at room temperature with gentle shaking (4050 rpm/min).
2) Delipidation with CUBIC-L3
1. Immerse the fixed sample in 10 mL of 0.5× CUBIC-L (1:1 dilution with water) in the 30 mL tube andincubate it for overnight at 37°C with gentle shaking (40-50 rpm/min).
2. Replace to 10-15 mL of 1× CUBIC-L in the 30 mL tube and delipidate for 3-5 days at 37°C with gentle
shaking (40-50 rpm/min).
■ Replace CUBIC-L every 2 to 3 days if the duration of treatment exceeds 3 days.
3. Wash the sample with 20 mL of PBS containing 0.05% NaN3 for 2 h × 3 times (or 2 h ×1, overnight ×1, 2h ×1) at 37°C with gentle shaking (40-50 rpm/min).
■ 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/0.05% NaN3 at 4°C at least for several months.
3) Cryosection of CUBIC-L-treated tissues
1. For cryopreservation, immerse the CUBIC-L-treated sample in 40% (w/v) sucrose/PBS and maintain it
at 4°C until the sample settles at the bottom of the tube (typically it takes over several hours to overnight).
2. Also, for cryopreservation of the fixed sample (without CUBIC-L treatment), immerse the sample in 10%(w/v) sucrose/PBS and keep at 4°C until the sample settles at the bottom of the tube. Subsequently,
exchange the reagent to 25% (w/v) and maintain it at 4°C until the sample settles again. Typically,
overnight incubation will be needed.
3. Embed the samples in O.C.T. compound.
4. Use a cryostat to prepare sections of these samples, cutting them at a thickness of 50 µm (for CUBICL-treated samples) or 30 µm (for fixed samples). Gather and wash the sections in PBS.
■ The prepared sections can be stored in PBS/0.05% NaN3 at 4°C for at least several months.

< Evaluate primary antibodies and HV-additives★ >STEP 1: Evaluate primary antibodies (2D staining)
In this step, the compatibility of primary antibodies with CUBIC-L-treated tissue samples is evaluated.
Cryosectioned tissue samples are stained with a selected primary antibody in the CUBIC-HV™2 Staining
buffer using a 24-well plate. It is advisable to compare the staining patterns between fixed tissue sections(without CUBIC-L treatment) and sections from CUBIC-L-treated samples, as the CUBIC-L treatment mayalter antigenicity. Additionally, the CUBIC-L treatment may have an antigen retrieval effect and occasionallyenhances the immunostained signals.
Procedures:
1. Calculate the required amount (X, Y) of primary and secondary (Fab fragment) antibodies.
■ A total of 250 µL of staining solution is used per well to stain a single slice in a 24-well plate.
■ Initiating the dilution rate of any primary antibodies at 1/100 or 1/1000 is recommended.
For example, when using the 1 µg/µL of primary antibody product and the 1.5 µg/µL of secondary Fabfragment product:
X (volume) = 2.5 µL of the primary antibody (1/100 dilution) /well
X (amount) = 2.5 (µL) × 1.0 (µg/µL) = 2.5 µg /well
Y (volume) = 2.5 (µg) / 1.5 (µg/µL) ≃ 1.67 µL of the secondary Fab fragment/well(primary : secondary = 1 : 1 in weight ratio)
2. Mix X µL of primary antibody and Y µL of secondary antibody in a Protein LoBind 500 µL tube. Incubate it
protected from light for 1.5 h at 37°C.
■ When using a direct dye-conjugated primary antibody, this can be skipped.
3. Preparation of the antibody staining solution.
For 1/100 dilution:
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
Distilled water
125 - (X + Y) µL
Antibody complex
X + Y µL (final 1/100-diluted primary antibody)=====================================
Total
250 µLFor 1/1000 dilution:
2× CUBIC-HV™2 Staining buffer
112.5 µL (final 1×)
Distilled water
112.5 µL
The above 1/100-diluted staining solution
25 µL*==================================================
Total
250 µL*Take a portion of the 1/100-diluted staining solution
4. Immerse a section in the antibody staining solution using a 24-well plate. Incubate it protected from lightfor 1 day at room temperature (approximately 25°C) with gentle shaking (40-50 rpm/min) using a table shaker.
■ Cover the wells with Parafilm to prevent evaporation of the antibody staining solution, and thenplace a lid on the 24-well plate.
5. Incubate it protected from light for 1 h at 4°C to stabilize the binding of the secondary Fab fragment.
6. During step 5, prepare 2 mL of 1× CUBIC-HV™2 Wash buffer with DAPI (1/1000) and cool it on ice.
■ For preparing 1× CUBIC-HV™2 Wash buffer, dilute 1 mL of 2× CUBIC-HV™2 Wash buffer in the kit
with the same amount of distilled water.
7. Wash the stained section in 1 mL of the ice-cold 1× CUBIC-HV™2 Wash buffer with DAPI in a 24 wellplate protected from light for 30 min x 2 times at 4°C with gentle shaking (40-50 rpm/min).
■ The wash buffer should be cooled at 4°C or on ice before use.
8. Replace the wash buffer to 1 mL of ice-cold PBS in a 24 well plate protected from light for 10 min at 4°C
with gentle shaking (40-50 rpm/min).
■ PBS should be cooled at 4°C or on ice before use.

9. Place the sections on a slide glass for preparation and observed it with an epifluorescence microscope.
■ For optimal assessment and comparison of staining signals, it is advisable to capture a complete
image of the preparation, incorporating all sections, utilizing a low-magnification objective (4X) andemploying the tiling function of the microscopy system.
-If an appropriate staining signal is obtained, proceed to STEP 2.
■ Choose an appropriate dilution rate (1/100 or 1/1000) based on the outcome.
-If no or only a weak signal is obtained, or non-specific signals are observed, follow thetroubleshooting as below:■ The fixation method, such as PFA concentration or fixation time, should be considered. Try reduced
PFA concentration (4% to 1 or 2%) and skip the post-fixation step.
■ CUBIC-HVTM does not include an antigen activation step since the CUBIC-L treatment may have acertain antigen-activating effect.
■ Staining at 4°C can occasionally improve the results.
■ If the above-mentioned optimizations fail to yield an acceptable result, the antibody should be
excluded.

STEP 2: Evaluate suitable HV-additives★ for each primary antibody
HV-additives★ are our patented probe penetration enhancers, each with distinct stringencies. Selecting anappropriate additive or combination of additives is essential for the successful 3D staining using CUBICHV™2. This section details the process of evaluating a suitable HV-additive★ for a specific primary antibody.
Recommended HV-additive★ combinations for the initial evaluation:1) No additive2) 0.5× Additive A3) 1× Additive A4) 1× Additive A + 1× Additive B (or 2× Additive B for further enhancement)5) 1× Additive B + 1× Additive C (or 2× Additive C for further enhancement)6) 1× Additive A + 1× Additive B + 1× Additive D7) 1× Additive B + 1× Additive C + 1× Additive D8) 1× Additive E9) 1× Additive F10) 1× Additive A + 1x Additive F■ Combinations 2) to 5) encompass most antibody cases. Users can initiate testing with these fiveconditions.
■ Additive D is used to further enhance antibody penetration in combinations 4), 5), and 10).
■ Combination 8) is recommended for antibodies exhibiting extreme resistance to penetration. Itshould be noted that the high stringency of this additive may significantly reduce antibody signals.
In such cases, testing a more diluted condition (e.g., 0.5x additive E) is advisable.
■ Combinations 9) and 10) serve as optional alternatives and can be compared to combinations 3) to
7).
Procedures:
1. Calculate the required amount (X, Y) of primary and secondary (Fab fragment) antibodies.
■ A total of 250 µL of staining solution is used per well to stain a single slice in a 24-well plate.
■ Use the dilution rate (1/100 or 1/1000) of the primary antibody determined in STEP 1.
For example, when using the 1 µg/µL of primary antibody product and the 1.5 µg/µL of secondary Fabfragment product at the dilution rate of 1/100:
X (volume) = 2.5 µL of the primary antibody (1/100 dilution) /well
X (amount) = 2.5 (µL) × 1.0 (µg/µL) = 2.5 µg /well
Y (volume) = 2.5 (µg) / 1.5 (µg/µL) ≃ 1.67 µL of the secondary Fab fragment/well(primary antibody: secondary Fab fragment = 1 : 1 in weight ratio)
In the case of dilution rate at 1/1000:
First, prepare the 1/10-diluted antibody solutions (for testing all the 10 HV-additive★ combinations).
■ Modify the following recipes based on the number of tested HV-additive★ combinations.
Primary antibody
3 µL
Distilled water
27 µL==================================Total
30 µL (0.1 µg/µL of the primary antibody)
Secondary Fab fragment
2 µL
Distilled water
18 µL==================================Total
20 µL (0.15 µg/µL of the secondary Fab fragment)
Then, calculate X and Y.
X (volume) = 2.5 µL of the 1/10-diluted primary antibody (final 1/1000 dilution) /well
X (amount) = 2.5 (µL) × 0.1 (µg/µL) = 0.25 µg /well
Y (volume) = 0.25 (µg) / 0.15 (µg/µL) ≃ 1.67 µL of the 1/10-diluted secondary Fab fragment /well(primary antibody : secondary Fab fragment = 1 : 1 in weight ratio)

2. To test all 10 combinations simultaneously, mix 11 times X µL of primary antibody and 11 times Y µL of
secondary antibody in a Protein LoBind 500 µL tube. Incubate the mixture protected from light for 1.5 h at
37°C.
■ Adapt the mixing volume based on the number of combinations being tested.
■ When using a direct dye-conjugated primary antibody, this step can be skipped.
3. Preparation of the antibody staining solutions 1)-10) containing HV-additive★.
1) No additive (control)
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
Distilled water
125 - (X+Y) µL
Antibody complex
X + Y µL=====================================
Total
250 µL2) 0.5x Additive A
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive A
12.5 µL (final 0.5×)
Distilled water
125 - (X+Y-12.5) µL
Antibody complex
X + Y µL=====================================
Total
250 µL3) 1x Additive A
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive A
25 µL (final 1×)
Distilled water
125 - (X+Y-25) µL
Antibody complex
X + Y µL=====================================
Total
250 µL4) 1x Additive A + 1x Additive B
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive A
25 µL (final 1×)
10× Additive B
25 µL (final 1×)
Distilled water
125 - (X+Y-50) µL
Antibody complex
X + Y µL=====================================
Total
250 µL■ Alternatively, 1× Additive A + 2× Additive B can be considered for further enhancement.
5) 1x Additive B + 1x Additive C
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive B
25 µL (final 1×)
10× Additive C
25 µL (final 1×)
Distilled water
125 - (X+Y-50) µL
Antibody complex
X + Y µL=====================================
Total
250 µL■ Alternatively, 1× Additive B + 2× Additive C can be considered for further enhancement.

6) 1x Additive A + 1x Additive B + 1x Additive D
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive A
25 µL (final 1×)
10× Additive B
25 µL (final 1×)
10× Additive D
25 µL (final 1×)
Distilled water
125 - (X+Y-75) µL
Antibody complex
X + Y µL=====================================
Total
250 µL7) 1x Additive B + 1x Additive C + 1x Additive D
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive B
25 µL (final 1×)
10× Additive C
25 µL (final 1×)
10× Additive D
25 µL (final 1×)
Distilled water
125 - (X+Y-75) µL
Antibody complex
X + Y µL=====================================
Total
250 µL8) 1x Additive E
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive E
25 µL (final 1×)
Distilled water
125 - (X+Y-25) µL
Antibody complex
X + Y µL=====================================
Total
250 µL■ Alternatively, a diluted condition (e.g., 0.5x Additive E) can be considered when the antibody
signals are significantly reduced.
9) 1x Additive F
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive F
25 µL (final 1×)
Distilled water
125 - (X+Y-25) µL
Antibody complex
X + Y µL=====================================
Total
250 µL10) 1x Additive A + 1x Additive F
2× CUBIC-HV™2 Staining buffer 125 µL (final 1×)
10× Additive A
25 µL (final 1×)
10× Additive F
25 µL (final 1×)
Distilled water
125 - (X+Y-50) µL
Antibody complex
X + Y µL=====================================
Total
250 µL
4. Immerse a section in each of the antibody staining solutions using a 24-well plate. Incubate it protectedfrom light for 1 day at room temperature (approximately 25°C) with gentle shaking (40-50 rpm/min) using atable shaker.
■ Cover the wells with Parafilm to prevent evaporation of the antibody staining solution, and thenplace a lid on the 24-well plate.
5. Incubate it protected from light for 1 h at 4°C to stabilize the binding of the secondary Fab fragment.
6. During step 5, prepare 2 mL of 1× CUBIC-HV™2 Wash buffer with DAPI (1/1000) and cool it on ice.
■ For preparing 1× CUBIC-HV™2 Wash buffer, dilute 1 mL of 2× CUBIC-HV™2 Wash buffer in the kit
with the same amount of distilled water.

7. Wash the stained section in 1 mL of the ice-cold 1× CUBIC-HV™2 Wash buffer with DAPI in a 24 wellplate protected from light for 30 min x 2 times at 4°C with gentle shaking (40-50 rpm/min).
■ The wash buffer should be cooled at 4°C or on ice before use.
8. Replace the wash buffer to 1 mL of ice-cold PBS in a 24 well plate protected from light for 10 min at 4°C
with gentle shaking (40-50 rpm/min).
■ PBS should be cooled at 4°C or on ice before use.
9. Place the sections on a slide glass for preparation and observed it with a fluorescence microscope.
■ For optimal assessment and comparison of staining signals, it is advisable to capture a complete
image of the preparation, incorporating all sections, utilizing a low-magnification objective (4X) andemploying the tiling function of the microscopy system.
10. Compare the staining signals in each of the tested HV-additive★ combinations with 1) No additive
condition.
-Omit the conditions that 1) causes higher background signals, an/or 2) significantly reduce thestaining signals.

< Determine the 3D staining condition for each primary antibody with HV-additives★ >STEP 3: Determine 3D staining condition for each primary antibody
During this final step, the compatibility of the chosen HV-additive★ combinations in STEP 2 for CUBIC-HVTM2
3D staining is assessed. HV-LLPS★ is also integrated into this process. It is advisable to utilize a relatively
small tissue block (e.g., a mouse brain hemisphere) for the evaluation. Once the optimal HV-additive★
combination is determined, the amount of the primary antibody is adjusted accordingly.
Procedures:■ The reagent volumes specified here are based on the usage of a mouse brain hemisphere (0.3 g).
Adjust the necessary volumes accordingly for other tested organs/tissues.
1. Immerse the delipidated sample (prepared through steps 1) and 2) of the above <Sample preparation>
procedure) in 5 mL of 1× CUBIC-HV™2 Pre-wash buffer in the 15 mL standing tube and incubate it forovernight at 37°C with gentle shaking (40-50 rpm/min).
2. Exchange the buffer to 5 mL of 1× CUBIC-HV™2 Staining buffer with or without selected 1× HV-additive★
reagents and incubate it for 1.5 h~ at 37°C with gentle shaking (40-50 rpm/min).
For 5 mL of the 1× CUBIC-HV™2 Staining buffer containing a single HV-additive★
(e.g., the HV-additive★ combination 3) in STEP 2):
2× CUBIC-HV™2 Staining buffer
2.5 mL (final 1×)
10× HV-additives★ reagent
0.5 mL (final 1×)
Distilled water
2 mL============================================================
Total
5 mL
■ Prepare all the staining buffer recipes containing the selected combinations of HV-additive★
identified in STEP 2.
As a control, 5 mL of the 1× CUBIC-HV™2 Staining buffer without HV-additive★ is required:
2× CUBIC-HV™2 Staining buffer
2.5 mL (final 1×)
Distilled water
2.5 mL============================================================
Total
5 mL
3. During the step 2, prepare the antibody mix as follows.
a. Calculate the required volume (X, Y) of the primary antibody and the secondary Fab fragment.
Primary antibody: 2.5 µg/mouse brain hemisphere (approximately 0.3 g)
To use 2.5 µg of 1 mg/mL (= 1 µg/µL) 1st Ab product, X = 2.5 (µg) /1 (µg/µL) = 2.5 µL.
Secondary Fab fragment: 2 µg/mouse brain hemisphere
In the case of using 1.5 mg/mL product, Y = 2 (µg)/1.5 (µg/µL) ≒ 1.33 µL.
■ When using a direct dye-conjugated primary antibody, 3-a can be skipped.
■ The quantity and final concentration of the primary antibody should be tailored to eachapplication (usually 5 µg/whole mouse brain at a concentration of 50-100 µg/mL). However, it isadvisable to begin by employing the indicated amount and concentration of antibody to initiallyassess HV-additive★ for 3D staining.
■ When applying to organs other than the brain, estimate the required quantity of antibodies andreagents by considering the weight ratio of the organ.

b. Prepare the following 3D staining solution (50 µL/mouse brain hemisphere).
For 50 µL of the 3D staining solution containing a single HV-additive★ reagent:
2× CUBIC-HV™2 Staining buffer
25 µL (final 1×)Primary antibody
X µL
Secondary Fab fragment
Y µL
10× HV-additives★ reagent
5 µL
Distilled water
25 - (X+Y+5) µL============================================================
Total
50 µL
Add the entire volume to the 500 µL Protein LoBind tube and incubate the mix for 1.5 h at 37°C.
■ The concentration of the primary antibody is 50 µg/mL (2.5 µg/50 µL) in this case.
■ Prepare all the staining buffer recipes containing the selected combinations of HV-additive★
identified in STEP 2.
As a control, 50 µL of the 3D staining solution without HV-additive★ is required:
2× CUBIC-HV™2 Staining buffer
25 µL (final 1×)Primary antibody
X µL
Secondary Fab fragment
Y µL
Distilled water
25 - (X+Y) µL============================================================
Total
50 µL
4. After the reaction in step 3, transfer the entire 3D staining solution into the 3D tissue staining pot[CSSR005].
■ This 3D tissue staining pot for CUBIC-HV™2 can be used repeatedly. After the usage, wash the
pot with a neutral detergent and distilled water.
5. Collect the sample with a metal spoon and put it into the staining pot.
■ A portion of the sample should be attached gently on soft paper towel to absorb residual buffer(see our general protocol for details).
6. Slowly pour CUBIC-HV™2 Wrapping reagent into the staining pot until the pot is filled. Remove thebubbles.
■ The wrapping reagent prevents samples from drying out during staining by covering the entire
sample and the staining solution containing condensed staining probes (HV-LLPS★).
7. Close the lid tightly and incubate the chamber for 3 days at RT with slow rotation (<1 rpm) under a lightshielded condition (see our general protocol for details).
8. To stabilize the secondary Fab signals, further incubate the staining chamber for 24 h at 4°C with slow
rotation (<1 rpm) under a light-shielded condition.
9. 1 h before moving to the step 10, prepare 1× CUBIC-HV™2 Wash buffer and cool it on ice.
For 20 (5 × 4) mL of the 1× CUBIC-HV™2 Wash buffer:
2× CUBIC-HV™2 Wash buffer
10 mL (final 1×)
Distilled water
10 mL============================================================
Total
20 mL
10. Recover the sample from the staining pot with a metal spoon. Discard the staining solution and thewrapping reagent.
11. Remove the residual buffer and the wrapping reagent as in step 5.
12. Immerse the sample in 5 mL of pre-cooled 1× CUBIC-HV™2 Wash buffer in the 15 mL standing tube.
Gently invert the tube several times to separate the wrapping reagent remaining on the sample surface.
13.Replace the 1× CUBIC-HV™2 wash buffer with a new one and continue washing the sample gently at
4°C with light shielding, while shaking at 40-50 rpm/min, for 2 h.
14. Replace the wash buffer with the new one and continue washing the sample two more times (2 h ×2).

15. During step 14, prepare ~4 mL of fixative solution by diluting formalin (FA) solution to final 1% in 1×
CUBIC-HV™2 Wash buffer and cool it on ice.
■ The saturated formalin solution contains 35 to 38% of formalin. For example, when using a 37%
formalin solution, dilute it with 1x CUBIC-HV™2 Wash buffer at a ratio of 1:36.
For 3.7 mL of the FA solution:
2× CUBIC-HV™2 Wash buffer
1.85 mL (final 1×)
Saturated formalin (37%)*
0.1 mL (final 1%)*The concentration may be different by products.
Distilled water
1.75 mL============================================================
Total
3.7 mL
16. Fix the sample in the 1% FA solution for 24 h at 4°C with gentle shaking (40-50 rpm/min) under a lightshielded condition.
17. To accelerate the fixation reaction, further incubate the sample for 1 h at 37°C with gentle shaking (4050 rpm/min) under a light-shielded condition.
■ Reducing the FA reaction time in steps 2 and 3 could result in the attenuation of antibody signals.
18. Wash the sample in 30 mL of PBS in the 50 mL tube for 2 h at RT with gentle shaking (40-50 rpm/min)under a light-shielded condition.
19. Evaluate antibody penetration by preparing cryosections from the central regions of the sample. Beginby immercing the 3D-stained and fixed sample in 40% (w/v) sucrose/PBS solution and keep it at 4°C
until the sample settles at the bottom of the tube (usually taking several hours to overnight) for
cryopreservation.
20. Embed the samples in O.C.T. compound.
21. Use a cryostat to prepare sections from the central regions of the samples, cutting them at a thicknessof 50 µm. Gather and wash the sections in PBS.
22. Place the sections on a slide glass for preparation and observed it with a fluorescence microscope.
■ For optimal assessment and comparison of staining signals, it is advisable to capture a complete
image of the preparation, incorporating all sections, utilizing a low-magnification objective (4X) andemploying the tiling function of the microscopy system.
23. Compare the staining signals in each of the tested HV-additive★ combinations with 1) No additive
condition.
-Select the best HV-additive★ combination according to the staining result.
-If required, try reducing the use of primary antibody amount with the determined HV-additive★
combination.

< Troubleshooting >1. insufficient penetration of antibody- extend the staining period- increase the antibody amount and concentration
2. High background signals or aggregated signals- reduce the antibody amount and concentration- test other HV-additive★ combinations
3. faint or absent staining signals- shorten the fixation period- reduce the PFA concentration- shorten the duration of CUBIC-L treatment- reduce the amount of HV-additive★
- test other HV-additive★ combinations

Reference
1. Susaki et al. in prep
2. Susaki et al. Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of
biological tissue. Nature Communications (2020) 11: 1982. DOI: 10.1038/s41467-020-15906-5
3. Matsumoto et al. Advanced CUBIC tissue clearing for whole-organ cell profiling. Nature Protocols(2019) 14: 3506–3537. DOI: 10.1038/s41596-019-0240-9
4. Susaki et al. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. Nature
Protocols (2015) 10: 1709–1727. DOI: 10.1038/nprot.2015.085