Comparison of tissue clearing achieved in rat brains processed with hydrophobic tissue clearing protocols. Comparison of clearing efficacies of Ethanol BABB (A), 1-propanol BABB (B), tert-butanol BABB (C), 3DISCO (D), iDISCO+ (E), uDISCO (F), FDISCO (G), vDISCO (H), Ethanol-ECi (I), and PEGASOS (J) in rat brain samples (3 mm thickness) with both PACA-Light and PACA-Glow. All tissue clearing time course are shown in Supplementary Figure S9. Optical images show samples after clearing (red dotted line), as well as any changes in sample size upon tissue processing (also Supplementary Figure S12). (K) Comparison of clearing efficacies of ten hydrophobic tissue clearing protocols. Three discs from three distinct brain regions (B1: prefrontal cortex and basal ganglia, B2: cerebral cortex and midbrain/diencephalon, B3: cerebellum) were generated and analyzed for each sample. Each color line and bar point to assessment values of empty (black) and refractive index matching solution (blue) of each protocol, and three distinct regions of the brain the B1 (square, red), B2 (diamond, violet), and B3 (triangle, sky blue). Green bars point to assessment values of glow light (unit) in dark. Results represent three replicates of each experiment, and data are presented as the mean ± SD (standard deviation). The transparency of the cleared brain was evident against a patterned background (length:width = 5 mm:5 mm).