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. 2020 May 29;6(22):eaba0365. doi: 10.1126/sciadv.aba0365

Fig. 1. A rapid method combining depigmentation with tissue clearing in representatives of four distinct and species-rich animal clades.

Fig. 1

(A) Main steps of the DEEP-Clear protocol, including incubation times for the five main model systems presented in this study. (B) Systematic advancement of eye depigmentation speed by acetone pretreatment in immature and mature worms. Quantified comparisons between acetone-treated (AcT) and untreated (UT) head halves incubated with Solution-1. All values are mean ± SD; statistical significance was determined by a Wilcoxon test, yielding P values of P = 0.00166 (immature worms) and P = 0.00192 (mature worms). (C) Systematic advancement of eye depigmentation speed by acetone pretreatment in squid. Quantification of depigmentation time in acetone-treated and untreated squid halves upon incubation with Solution- 1.1. Values are mean ± SD; statistical significance was determined by a Wilcoxon test (P = 0.01285). (D) Differential and synergistic impact of acetone, peroxide, and Solution-1.1 on zebrafish fin pigments. Panels show fins of untreated (top) and treated (bottom) zebrafish fins. Insets: Magnification of dashed area and impact of different treatments on respective pigments (black arrows). Xanthophore containing pteridine and carotenoid pigments (yellow and orange) and melanophore containing melanin pigment (black). Rightmost panels show the overall impact of the full DEEP-Clear protocol. (E) Wide-field images of specimens placed on top of a USAF 1951 chart. Uncleared samples in PBS (top panels), same samples after depigmentation and refractive index (RI) matching in Solution-2 (middle panels), and higher magnification of red rectangular areas indicating the highest level of transparency reached after RI matching (bottom panels). Scale bars in the insets of (D), 20 μm. In (A), dagger indicates the possibility of fixation with Bouin’s solution; asterisks indicate the use of Solution-1.1 incubation instead of Solution-1. o.n., overnight; RT, room temperature; h, hour; ’, minutes. In (B) and (C), *P < 0.05 and **P < 0.01. Photo credit: Marko Pende, Medical University of Vienna.