Fig. 1: Digital spatial proteomic profiling (DSP) using immunofluorescence as an indicator of multiple proteomic targets on a single slide.

A Working procedure of NanoString GeoMx Digital Spatial Profiling using nCounter MAX/FLEX. Paraformaldehyde-fixed cortical human tissue is incubated with 3 antibody visualization markers, followed by incubation with 75 DSP probes. After tissue is imaged, regions of interest (ROI) are digitally selected, ROIs are then subjected to ultraviolet (UV) light which results in release of UV-cleavable protein specific oligonucleotide barcodes from DSP probes. Cleaved protein unique barcodes are then collected and quantified. B Microphotograph of cortical tissue labeled with Iba1 (red), GFAP (green), and DNA (blue) visualization markers. Regions of interest (ROI) were manually drawn in approximately the same location within white matter, molecular layer (cortical layer I), and internal/granular layer (cortical layer IV) for every case. Four ROIs per subregion per patient (12 ROIs per tissue section) were collected and assessed as individual data points in all downstream analyses. Individual case demographics and cognitive status may be found in Supplementary Table 5. C DSP human Neuroscience protein probes, separated by disease or functional genre, a (*) indicates a protein that was not part of the NanoString Human Neuroscience panel but rather was chosen by our lab and spiked in.