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. Author manuscript; available in PMC: 2022 Jan 15.
Published in final edited form as: Mol Psychiatry. 2020 Jul 10;26(10):5766–5788. doi: 10.1038/s41380-020-0806-5

Fig. 7. Amyloid and Tau pathology are shown with six different methods in T21C5Δ7 organoids that have BACE2 copy number reduced from three to two by CRISPR/Cas9.

Fig. 7

a–d The signal of amyloid specific antibodies Aβx-40 + Aβx-42 (a, b), or 4G8 (c, d) colocalising with Thioflavine S in T21C5Δ7 (96DIV) organoids was drastically increased upon treatment with 87% Formic acid for 10 minutes at RT, proving it contains the insoluble extracellular β-amyloid deposits. Scale bar: 10 μm. e AT8 (hyperphosphorylated Tau) positive neurites within plaque-like structure in 48DIV organoids. Left: the whole organoid slice, scale bar: 500 μm. Right: zoom in on the plaque-like structure from ‘e’, in the three individual z-slices (interval between slices, 1 μm; scale bar: 20 μm). f AT8 (hyperphosphorylated Tau) positive neurites in 96DIV organoids. Scale bar: 10 μm. g–j TG3 (conformationally altered Tau) staining of unedited control T21C5 (100DIV) g, CRISPR-edited T21C5Δ7 (48DIV) with TG3-positive neurons in 48DIV organoids h, i, and CRISPR-edited T21C5Δ7 (96DIV) showing many TG3+ neurons with diffuse staining of extracellularized mal-conformed Tau aggregates j. Scale bar: 50 μm. k–n Gallyas staining of human AD brain k, unedited control T21C5 (100DIV) l, CRISPR-edited T21C5Δ7 (96DIV) m, n shows negative staining in parental unedited organoid l and very strong signal in neurons and plaque-like associated neurites within T21C5Δ7 organoid m, n. Scale bars: 50 μm l, m and 20 μm n and 5 μm k. o Representative western blot of T21C5 and T21C5Δ7 organoid lysates stained using antibodies against pathologically conformationally altered Tau (TG3) or general 3 repeat (3 R) Tau. β-actin was used as a loading control. Human brain tissue of a 75 year old is shown for comparison. Comparison of the average values (n = 4) for CRISPR-edited T21C5Δ7 showed a highly significant relative increase in TG3 compared with unedited (n = 4) T21C5 organoids, as indicated in the graph, p = 0.0127.