Organoids were treated for 30 or 60 days with a combination of beta secretase (BACE-1) inhibitor (β-Secretase Inhibitor IV, EMD Millipore) and gamma secretases inhibitor (Compound E; EMD Millipore) or equivalent DMSO vehicle. Each compound was diluted into culture medium from a 5 mM DMSO stock that had been stored at -20°C. The concentrations of BACE-1 and Comp-E that were used were 5 μM and 6 nM or 1 μM and 3 nM, respectively. Vehicle-treated cultures received the same concentration of DMSO. Drug treatment was begun at day 30 of the culture and maintained until the endpoint at 60 or 90 days of culture. Media containing treatment or vehicle was replaced every 4 to 5 days. Organoids were processed for immunohistochemistry as described above.
