N buthyldiethanolamine

The CUBIC-cancer protocol was followed [31 (link)]. Briefly, CUBIC-L2 solution (L2), for delipidation and decolourisation, was prepared as a mixture of 10 w%/10 w% Triton X-100 (Merck, Darmstadt, Germany)/N-buthyldiethanolamine (B0725 Tokyo Chemical Industry, Oxford, UK). CUBIC-R2 solution (R2), for RI matching (RI = 1.52), was prepared as a mixture of 30% (w/v) nicotinamide (Merck, Darmstadt, Germany) and 45% (w/v) antipyrine (Merck, Darmstadt, Germany).
For whole-organ clearing, 4% PFA fixed lungs were washed with PBS for 2 h, three times each, followed by immersion in CUBIC-L1 solution (L1) (50% (v/v) mixture of water and CUBIC-L2) for 6 h at 37 °C. Then, the organs were immersed in L2 solution at 37 °C for 48 h. The L2 solution was refreshed after 24 h during this process. After decolourisation and lipid clearing, the organs were washed with PBS at room temperature for 2 h, followed by immersion in CUBIC-R1 solution (R1) (50% (v/v) mixture of water and CUBIC-R2) for 6 h at room temperature. Finally, organs were immersed and stored in R2 solution at room temperature overnight.

CUBIC clearing solutions were prepared as follows^{19 (link)}. CUBIC-L solution for decolorization and delipidation was prepared as a mixture of 10% (w/w) polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 (Nacalai Tesque, Kyoto, Japan) and 10% (w/w) N-buthyldiethanolamine (Tokyo Chemical Industry, Tokyo, Japan). CUBIC-R solution for RI matching was prepared as a mixture of 45% (w/w) 2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine and 30% (w/w) nicotinamide (Tokyo Chemical Industry). Liver tissues were fixed by perfusion of 4% paraformaldehyde (PFA) via the left ventricle of the heart, and tissues were post-fixed in 4% PFA at 4 °C. After washing in phosphate-buffered saline (PBS), the fixed liver tissues were immersed in 50% CUBIC-L at 37 °C for more than 6 h followed by 100% CUBIC-R for 3–5 days. After washing in PBS, tissues were immersed in 50% CUBIC-R for 6 h followed by 100% CUBIC-R for more than 1 day. Whole-liver tissue images were acquired with a custom-built light-sheet fluorescence microscope (LSFM; developed by Olympus, Tokyo, Japan). Laser bandwidths of 488 nm and 532 nm/590 nm were used to detect Venus and tdTomato, respectively. Three-dimensionally rendered images were visualized, captured, and analyzed with the Imaris software program (ver 8.4, Bitplane AG, Zurich, Switzerland) and Free Imaris Viewer (ver 9.5, Bitplane AG).