To optically clear fixed fused spheroids, we adapted the iDISCO protocol described by Renier et al48. Briefly, after fixation with 4% PFA for 3 hrs, spheroids were dehydrated with a day-long methanol (MetOH) dilution series (20% to 100% MetOH). Next, they were incubated in 5% H2O2 overnight at 4°C. The following day, they were rehydrated with a reverse MetOH dilution series and incubated overnight in 0.2% Triton-X, 20% DMSO, 0.3 M Glycine/PBS at 37°C. The spheroids were then blocked with 0.2% Triton-X, 10% DMSO, 6% goat serum/PBS at 37°C for 2 days, followed by a heparin treatment for 2 hrs (PTwH: 0.2% Tween-20, 10 μg/mL Heparin/PBS) to reduce non-specific antibody binding. They were next incubated with a chicken anti–GFP (1:1500; GeneTex: GTX13970) antibody for 2 days in PTwH with 5% DMSO and 3% goat serum at 37°C. After a day-long wash series with PTwH, a secondary antibody diluted in PTwH, 3% goat serum was added for an additional two days at 37°C. After 2 days of PTwH washes, the spheroids were cleared by a three-step tetrahydrofuran (THF) series (80%, 100%,
