Cubic l

After fixation in 4% PFA, samples of CUBIC-L/R were immersed in 50 and 100% CUBIC-L (Tokyo Chemical Industry Co., Ltd., Tyoko, Japan) at 37°C for 72 h in a shaking incubator. Samples were washed in PBS for 24 h and pre-treated in 50% CUBIC-R+ (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) at room temperature for 24 h. Samples were then incubated in 100% CUBIC-R+ solution at room temperature for 48 h. Sample of CUBIC-X were immersed in 50 and 100% CUBIC-L at 37°C for 5 days in a shaking incubator. Samples were washed in 0.1 M PBS for 24 h and treated in CUBIC-X1 (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) solution (20% imidazole in dH₂O) at 4°C for 60 h. Samples were then incubated in CUBIC-X2 (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) solution (5% imidazole and 55% antipyrine in dH₂O) at room temperature for 36 h.

D1-tdTomato and D2-tdTomato mice were anesthetized with isoflurane (3%, inhalation) and transcardially perfused with 20 mL of PBS containing 10 U/mL heparin and then with 4% paraformaldehyde (PFA; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) in 0.1 M phosphate buffer (PB, pH 7.4). The excised brain was post-fixed with the same fixative for 18–24 h at 4 °C. After being washed with PBS for 6 h (2 h × 3), the brain was immersed in PBS-diluted CUBIC-L (1:1) (Tokyo Chemical Industry Co., Ltd, Tokyo, Japan) and shaken gently (60 rpm, RT) for at least 8 h. On the next day, the brain was transferred to undiluted CUBIC-L and shaken overnight (60 rpm, RT). For whole-brain delipidation, the brain was immersed in fresh CUBIC-L and shaken gently (60 rpm, 37 °C) for 4 days. The CUBIC-L was changed every 1–2 days. On days 7 and 8, the brain was immersed and shaken overnight using PBS and PBS-diluted CUBIC-R⁺ (1:1) (Tokyo Chemical Industry), respectively. Finally, the brain was transferred to undiluted CUBIC-R⁺ and shaken gently (60 rpm, RT). Image-acquisition was performed using an UltraMicroscope II (Zoom body configuration) (Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany). 3D imaging analysis was conducted using an Imaris (Oxford Instruments, Abingdon, UK).

The mice were sacrificed with CO₂ gas and washed with PBS under physiological pressure. The lungs were placed in 4% paraformaldehyde for 2 h, followed by extensive washing with PBS. Subsequently, the lungs were permeabilized with 1% Triton X-100 (St. Louis, MO, USA) in PBS containing 2% skim milk, followed by overnight incubation with an anti-Tuj1 antibody. The lungs were washed thrice with PBS containing 0.2% Tween 20 for 10 min. The lungs were fixed again with 4% paraformaldehyde for 10 min after Alexa555-conjugated anti-rabbit IgG incubation and then placed in CUBIC-L and CUBIC-R solutions to make the lungs transparent (Tokyo Chemical Industry). The transparent lungs were observed, and Z-stack images were obtained using an LSM710 confocal microscope (Zeiss, Oberkochen, Germany) or a stereoscopic microscope (LEICA Microsystems, Wetzlar, Germany).

For 3D reconstruction imaging, the aforementioned 96-well plate comPDAC-FPCL models were employed. The models were sequentially submersed in CUBIC-L and CUBIC-R (T3740 and T3741, respectively; Tokyo Chemical Industry CO., LTD., Tokyo, Japan), according to the manufacturer’s instructions, for staining pretreatment. Thereafter, the models were stained with vimentin (1:200) and EpCAM (1:200) antibodies, followed by the corresponding secondary antibodies. Finally, the models were transferred to glass-bottom culture dishes and infiltrated using a mounting solution (RI 1.520). The 3D reconstruction images were captured using a Zeiss confocal microscope (LSM 700; Oberkochen, Germany).

CUBIC-L/R tissue clearing was performed according to the previous literature^{53 (link),54 (link)}. In brief, a fixed whole brain of Thy1-YFP-H Tg mouse was treated with the commercialized CUBIC-L (Tokyo Chemical Industry, Japan, #T3740) at 37 °C for 4 days. The delipidated and PBS-washed sample was then stained with propidium iodide (PI) for counterstaining by immersing in HEPES-NaCl buffer (10 mM HEPES: TCI, #H0396; 500 mM NaCl: TCI, #S0572; 0.05% NaN₃: nacalai tesque, #31208-82) with 3 µg/mL PI (DOJINDO, Kumamoto, Japan, #343-07461) at 37 °C for 5 days. After PBS washing, the sample was RI matched with commercialized CUBIC-R + (N) (TCI #T3983) and then embedded in CUBIC-R-agarose as in the previous reports^{54 (link)}.

To clear the brain block samples containing cortical arteries using the CUBIC method, CUBIC reagents were prepared according to previous reports [62 (link),63 (link)]. The reagent CUBIC-L (T3740, Tokyo Chemical Industry, Tokyo, Japan), used for decoloring and delipidation, comprises 10 wt% N-butyldiethanolamine and 10 wt% Triton X-100 in distilled water. The reagent CUBIC-R (Tokyo Chemical Industry), used for clearing, comprises 45 wt% antipyrine (D1876, Tokyo Chemical Industry) and 30 wt% nicotinamide (N0078, Tokyo Chemical Industry). The pH 8–9 was adjusted by N-butyldiethanolamine (B0725, Tokyo Chemical Industry) in distilled water.