1 3 dimethyl 2 imidazolidinone

UbasM-1 (Ub-1) was prepared as a mixture of 25 wt% Meglumine (Shanghai Macklin Biochemical Co., M813277), 25 wt% Urea (Sigma-Aldrich, 15604), 20 wt% 1,3-Dimethyl-2-imidazolidinone (Sigma-Aldrich, cat. no. 40727), and 0.2 wt% Triton X-100 (Sigma-Aldrich, 10789704001). UbasM-2 (Ub-2) was prepared as a mixture of 25 wt% Urea (Sigma-Aldrich, 15604), 20 wt% 1,3-Dimethyl-2-imidazolidinone (Sigma-Aldrich, 40727), and 40 wt% Sucrose (Sigma-Aldrich, S9378). For brain slice clearing, each fixed slice was immersed in 5–10 ml of Ub-1 at room temperature for 1–2 hours. For whole organ clearing, each fixed sample was immersed in 10–20 ml of Ub-1 at 30 °C for 3–5 days with gentle shaking. The treated sample was then washed with PBS several times and then stored at 4 °C for 12 hours. Then the washed sample was immersed into in 10–20 ml of Ub-2 at 30 °C for 3–5 days. After imaging, the sample was again washed with PBS, immersed in 20% (w/v) Sucrose in PBS, and stocked in O.C.T. compound at –80 °C. Agarose-embedded samples typically required longer incubation times for efficient clearing. We recommend embedding the samples in agarose after Ub-1 clearing and PBS washing and minimizing the size to allow efficient penetration of Ub-2. Penetration efficiency can also be improved by clearing tissues at 37 °C but with larger size expansion, less firmness and partial quenching of fluorescent proteins.

Embryos were dissected in FHM medium (95 mM NaCl; 2.5 mM KCl; 0.35 mM KH₂PO₄; 0.2 mM MgSO₄.7H2O; 10 mM Na lactate; 0.2 mM Na pyruvate; 0.2 mM Glucose; 1.0 mM Glutamine; 0.01 mM EDTA; 4.0 mM NaHCO₃; 1.71 mM CaCl₂.2H₂O; 20 mM HEPES; pH adjusted to pH 7.4; after which 1 g/L BSA and 1x Pen/strep (Cat# GIBCO 15140148) were added).
For imaging with the light sheet MuVi SPIM, embryos were fixed in PBS with 4% PFA (ThermoScientific, Cat# 28908) for 10 min at room temperature (short fixation time to reduce background), mounted in 2% LMP agarose (Thermo Scientific, Cat# R0801) in PBS, and imaged immediately.
For imaging with the light sheet UltraMicroscope or OPT, embryos were fixed in PBS with 4% PFA overnight at 4 °C. They were cleared in UBAS-M (25 wt% Meglumine (Sigma-Aldrich, Cat# M9179), 25 wt% Urea (Sigma-Aldrich, Cat# 15604), 20 wt% 1,3-Dimethyl-2-imidazolidinone (Sigma-Aldrich, Cat# 40727), and 0.2 wt% Triton X-100 (Sigma-Aldrich, Cat# 10789704001)) [43 (link)] in 50 ml per embryo overnight at RT and mounted in 4% LMP agarose in UBAS-M before imaging.