Cubic r

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).

Cholesteatoma model mice were perfused with 4% PFA, and dissected parietal bone tissues were further fixed with 4% PFA overnight at 4 °C followed by decalcification for 7 days in CUBIC-B (Tokyo Chemical Industry, Tokyo, Japan). After decalcification, samples were cut manually under a microscope along the well-defined mass lesions on the parietal bones. Samples were stained with 100 µM Alexa Fluor 488 NHS ester (A20000; Thermo Fisher Scientific) for 1 h at ambient temperature, followed by washing three times with PBS. After staining, samples were treated for 15 min at 37 °C with CUBIC-R (Tokyo Chemical Industry). After refractive index (RI) adjustment, the samples were placed in a chamber with the same RI matching solution, coverslipped, and imaged by confocal microscopy (A1; Nikon).

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).

Staining and optical clearing of dissected tracheae were performed as described in a previous study (Hirashima and Adachi, 2015 (link)). Briefly, the samples were fixed with 4% PFA in PBS overnight at 4°C. For anti-SOX9 staining, the samples were incubated in 25 mg/mL hyaluronidase (Nacalai Tesque, #18240-36) for 1 h at 37°C, to digest hyaluronic acid. The samples were then blocked in 10% normal goat serum (Abcam, #ab156046) diluted in 0.1% Triton X-100/PBS (PBT) for 3 h at 37°C. The samples were treated with primary antibodies overnight at 4°C, washed in 0.1% PBT, and subsequently incubated in secondary antibodies conjugated to either Alexa Fluor 546 or Alexa Fluor 647 overnight at 4°C. DAPI was used for nuclear counterstaining (Dojindo Molecular Technologies, #D523-10, 1:200 dilution). The samples were mounted with 10 μL of 1% agarose gel onto a glass dish (Greiner Bio-One, #627871) for stable imaging. Then, the samples were immersed in CUBIC-R+ (Tokyo Chemical Industry Co., # T3741) solution for optical clearing. Images were acquired using the confocal laser scanning platform Leica TCS SP8 equipped with the hybrid detector Leica HyD, using a ×40 objective lens (NA = 1.3, WD = 240 μm, HC PL APO CS2, Leica) and the Olympus FluoView FV1000 with a ×30 objective lens (NA = 1.05, WD = 0.8 mm, UPLSAPO30XS, Olympus).

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.