D sorbitol

ScalesSQ tissue clearing was performed as previously described by Hama et al., 2015 (link). Briefly, we incubated organoids in ScaleSQ (22.5% (w/v)) D-sorbitol (Sigma) and 9.1 M urea (Sigma) for 2 hr at 37°C. Next, we exchanged the solution to ScaleS4 (40% (w/v)) D-sorbitol, 10% (w/v) glycerine (Roth), and 15–25% (v/v) DMSO (Sigma) also at 37°C. ScalseS4 was renewed after 2 hr and the organoids were maintained at 37°C until analysis. This is critical, as the clearing effect decreases visibly after 1 hr at room temperature.

NaCl (Carl Roth), ZnCl₂ (Carl Roth), AgNO₃ (Carl Roth), HgCl₂ (Carl Roth), NiCl₂ (Applichem), AsNaO₂ (Sigma Aldrich), NiCl₂ x 6 H₂O (Sigma Aldrich), CdSO₄ x 8/3 H₂O (Sigma-Aldrich), CuSO₄ (Sigma Aldrich), Pb(NO₃)₂ (Carl Roth) and guanidine hydrochloride (Carl Roth), respectively D-Sorbitol (Carl Roth) were dissolved to a concentration of 1 M, stored at—20°C and diluted in DMEM complete. For continuous treatment, the medium in the well was removed and replaced with inducer, diluted in DMEM complete for 6 h or 24 h. For 1 h induction cells were induced for 1 h with DMEM complete containing heavy metals. After 1 h this medium was removed and replaced fresh DMEM complete.