Tissue clearing – CUBIC1

PBS in the well plate was replaced with a mixture of 1:1 CUBIC1 solution and distilled water plus Hoechst stain (Life Technologies – H3570, 1:2000, 1ml per well). The seam of the well plate was sealed with Parafilm and the well plate was placed in a shaking waterbath at 37°C for 3h. The CUBIC1/water solution was then discarded and replaced with 100% CUBIC1 solution plus Hoechst and kept at 37°C overnight. The following day, the samples were washed in PBS three times for 1h on a shaker. The samples were then placed in 30% sucrose in PBS until the sections sank to the bottom of the wells. After, the sections were immersed in OCT and frozen at −80°C at least overnight.