Fig 1: High-plex 4i tissue profiling of human postmortem spinal cord. Fresh frozen tissue sections from ALS patient spinal cords were subjected to 14-plex profiling over six 4i cycles. (A) K-means clustering of 4i data was used to visualize tissue domains defined by all 14 channels across the entire tissue section. Each of 7 clusters is assigned a unique color and inset locations are noted (B–E). (B') Higher magnification image of anterior horn inset region (B). Cell type marker MBP (cyan) is expressed by oligodendrocytes, MAP2 (magenta) is expressed in neurons and neuronal processes, and ELAVL3 (yellow) is expressed by neurons, including motor neurons. (B?) Same as (B'), but yellow is used to visualize tissue autofluorescence. (B'?) Pathological markers P62 (cyan), TDP-43 (magenta), and hyperphosphorylated TDP43 (yellow) are visualized in the same FOV as in (B',B?). (C) Higher magnification image of lateral white matter inset region (C). Cell type markers NFH expressed in neurons (cyan), GFAP expressed in astrocytes (magenta), and CD68 expressed in activated microglia (yellow) are visualized. (D) Higher magnification image of ventral white matter inset region (D). Cell type markers NFH expressed in neurons (cyan), IBA1 expressed in microglia (yellow), and nuclear envelope marker LMNB1 (magenta), are visualized. (E) Higher magnification image of dorsal horn inset region (E). Cell type markers CD34 expressed in endothelial cells (cyan), ALDH1L1 expressed in astrocytes (yellow), and nuclear envelope marker LMNB1 (magenta), are visualized.