containing 5% horse serum and Hoechst (for nuclear immunoreactivity, source) and sections were incubated in secondary for 1 hour at room temperature before washing as before. Sections were then coverslipped in Fluoromount-G (Electron Microscopy Sciences, Hatfield, PA) mounting medium and the edges sealed with nail polish. For consistency, sections from control (Ctrl) and familial Alzheimer’s disease (fAD) organoids were processed in the same batch. The primary antibodies used with following dilutions: Microtubule-associated protein 2, MAP2 (chicken/1:400, Biolegend, San Diego, CA), 4G8 (mouse/1:400, Biolegend, San Diego, CA), β-amyloid (D54D2) (rabbit/1:400, Cell Signaling Technology, Danvers, MA), phosphorylated Tau (Ser396 –PHF13 and Thr181—D9F4G) (mouse and rabbit/1:400, Cell Signaling Technology, Danvers, MA), Early endosome antigen 1 (EEA1) (mouse/1:500, BD Biosciences, San Jose, CA) and Cleaved Caspase-3 (Asp175) (rabbit, 1:500, Cell Signaling Technology, Danvers, MA). Secondary anti-mouse, anti-rabbit, and anti-chicken antibodies conjugated to cyanine dyes (Cy2, Cy3 and Cy5, respectively) were purchased from Jackson Immuno Research laboratories (West Grove, PA). Twenty-four hours after coverslipping, sections were imaged using a laser scan confocal microscope (LSM710, Carl Zeiss) by researchers blind to experimental condition. For each tissue section and organoid, we imaged five different regions. Each treatment and conditioned contained between five and eight organoids.
