D1876

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.

Clearing cocktails were composed of five chemicals, which were selected by chemical screening^{24 (link)}. CUBIC-P− for decolorization was prepared as a mixture of 10 wt% 1-methylimidazole (M0508, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). CUBIC-L for decolorization and delipidation was prepared as a mixture of 10 wt% polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 (12967-45, nacalai tesque, Kyoto, Japan) and 10 wt% N-butyldiethanolamine (B0725, Tokyo Chemical Industry Co., Ltd.). CUBIC-R+ for RI adjustment was prepared as a mixture of 45 wt% 2,3-dimethyl-1-phenyl-5-pyrazolone/antipyrine (D1876, Tokyo Chemical Industry Co., Ltd.), 30 wt% nicotinamide (N0078, Tokyo Chemical Industry Co., Ltd.), and 0.5 wt% N-butyldiethanolamine.