In situ sequencing of immature and mature engram neurons. (A) A scheme of the in situ sequencing workflow. (B–E) Representative images of individual gene expression readout following sequencing and imaging. Slc17a6 (B), Tpbg (C), Unc5c (D), and Vipr2 (E). Scale conversion for individual images = 0.32 μm/pixel. (F–H) Cell segmentation. An image of DAPI-stained nuclei is shown with the estimated cell border boundary overlaid. (I–L) Representative example of DAPI (I), NeuN (J), eYFP⁺NeuN⁺Egr-1⁺DAPI⁺ neuron (L, white arrowhead)–stained cells that underwent in situ sequencing. (M)t-SNE plots of the in situ sequencing data. Points represent 50 μm²–binned areas covering the entire section. Areas in the DG for C-NB, C-NBF, and T-NBF are shown in blue, orange, and green, respectively, above other areas (gray). Scale bar = 1,000 μm (B–F); 225 μm (G); 55 μm (H); 175 μm (I–K); 75 μm (L).