Fig. 7

Validation of identified representative DEGs in layers II/III/V from AD and CT at the single-cell level using RNAscope smFISH. (A) Representative four rounds of whole-tiles RNAscope images were registered with their corresponding post-Immunofluorescence (IF) staining of Aβ, AT8, and GFAP (left panel of each quadrant). The representative high-mag images of layers II/III and V (left panel of each quadrant) were selected from the regions in the whole-tiles images circled by red (layer II/II) and green (layer V) rectangles. Individual cell was labeled as microglia (MG), astrocyte (AG), neuron (Neu), or oligodendrocyte (Oligo) according to its P2RY12/C1QB, GFAP, RBFOX3, and MBP expression respectively. (B) Stacked bar plots show the quantification of RNAscope probes against human SLC1A3, KIF5A, SNCG, STMN2, CSRP1, PLP1, GLUL, PAQR6, CD9, C1QB, SPP1, CD63, CRYAB, and YWHAH in microglia (P2RY12⁺/C1QB⁺), astrocytes (GFAP⁺), neurons (RBFOX3⁺), and oligodendrocytes (MBP⁺). RNA counts per cell were compared between three CT and three AD cases. For quantification, we identified 20–50 microglia, astrocytes, and oligodendrocytes, and 60–90 neurons from each case. Detailed information regarding quantification can be found in the methods section. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Mann–Whitney test, CT vs AD). Scale bar, 60 µm for layers II/II and V high-mag images, 360 µm for whole-tiles. See high-resolution RNAscope images at: https://bmbls.bmi.osumc.edu/scread/stofad-2