FDISCO clearing consisted of two steps: dehydration and refractive index matching. Tissues were dehydrated with THF solutions (mixed with dH2O, pH adjusted to 9.0 with triethylamine) at a series of concentrations 50, 70, 80, and 100 volume % (twice or thrice). Pure DBE (108014, Sigma-Aldrich) was used as a refractive index matching solution to clear tissue after dehydration. All steps were performed at 4°C with slight shaking. During clearing, the tissues were placed in glass chambers covered with aluminum foil in the dark. The incubation time of each step depended on the tissue type and size (table S1).

For bone clearing, mouse tibias were decalcified with 0.1 M EDTA-2Na (dissolved in 0.01 M PBS) for 2 to 3 days at 37°C with slight shaking and then washed with PBS several times prior to THF treatment. For whole-body clearing, mouse hair was removed before perfusion, and the whole body was washed with PBS several times at 37°C to remove residual blood after PFA fixation.

After FDISCO clearing, the tissues were stored in DBE in airtight glass chambers at 4°C in the dark. As tissue transparency might decrease during long-term storage after clearing, prior to repeated imaging, the tissues should be transferred to 100 volume % THF for several hours and then incubated again in DBE until the tissues were transparent. The peroxides in THF and DBE were removed by column absorption chromatography with basic activated aluminum oxide (20001861, Sinopharm Chemical Reagent Co. Ltd., China) (24). The clearing agents were freshly prepared. All other clearing protocols, including BABB, 3DISCO, uDISCO, FluoClearBABB, Ethanol-ECi, CUBIC, and PACT, were performed following the original papers (16, 17, 22, 23, 2628).

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