Neurons derived from the APPDp1-1 line had previously exhibited increased levels of secreted Aβ40 and Aβ42 (Israel et al., 2012). To determine whether this phenotype was perpetuated in 3D culture, we subjected culture media from fAD and control organoids to ELISA. In agreement with the previous study, we detected significantly higher levels of Aβ in the media from fAD organoids culture compared to controls (Fig 1A). We performed immunohistochemistry on fixed cryosections from control and fAD organoids to examine AD-like pathology using two different antibodies that recognize Aβ. The first antibody, 4G8 (immunoreactive against amino acid residues 17–24 of Aβ) has been widely used to label both soluble and aggregated Aβ [68]. In addition, we co-labeled sections with the anti-Aβ antibody D54D2, which recognizes several isoforms of amyloid (Aβ37, Aβ38, Aβ39, Aβ40, and Aβ42). In 90d organoids, we detected aggregates that were immunopositive for both Aβ antibodies (Fig 1B), while 4G8 immunoreactivity appeared against intracellular amyloid that co-localizes with MAP2 as well as putative extracellular aggregates (Fig 1B, S4A Fig and S1 and S2 Movies). We then used the D54D2
