Spinal Cord Clearing via FDISCO Protocol

The FDISCO protocol was performed according to the original literature (Qi et al., 2019 (link)). Before clearing, tetrahydrofuran (THF, Sigma-Aldrich, St. Louis, MO, United States) and dibenzyl ether (DBE, Sigma-Aldrich, St. Louis, MO, United States) were preprocessed by column absorption chromatography with basic activated aluminum oxide (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) to remove the peroxides. The spinal cords were dehydrated in THF solutions diluted in dH₂O (pH adjusted to 9.0−9.5 with trimethylamine; Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at graded concentrations as follows: 50, 70, 80, and 100 vol% (twice) for 45−60 min at each step. After dehydration, pure DBE was used as a refractive index matching solution to clear the spinal cord. During the clearing process, the spinal cord was placed in a glass chamber under the dark. All steps were performed at 4°C with slight shaking.