Protocol for spatial profiling of immune cells in the mouse cornea and conjunctiva using NanoString GeoMx DSP and nCounterPro platforms

Quantification of immune cells and their activation status in a spatially specific manner in a healthy or diseased tissue is a valuable tool in immunology. Here, we present a protocol for the spatial profiling of immune cells in the mouse cornea and conjunctiva. We describe steps for immunostaining with photocleavable oligonucleotide-conjugated antibodies, selecting regions of interest (ROIs), and collecting UV-cleaved oligonucleotides using GeoMx DSP. We then detail procedures for hybridizing collected oligonucleotides, cartridge blotting using nCounter Pro Prep, data acquisition, and data analysis. • Nuclear and oligonucleotide-conjugated antibody staining of mouse eye tissue sections • GeoMx DSP instrument for region-of-interest selection and oligonucleotide collection • Oligonucleotide hybridization, blotting on cartridge, cartridge imaging, and data analysis Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Quantification of immune cells and their activation status in a spatially specific manner in a healthy or diseased tissue is a valuable tool in immunology. Here, we present a protocol for the spatial profiling of immune cells in the mouse cornea and conjunctiva. We describe steps for immunostaining with photocleavable oligonucleotide-conjugated antibodies, selecting regions of interest (ROIs), and collecting UV-cleaved oligonucleotides using GeoMx DSP. We then detail procedures for hybridizing collected oligonucleotides, cartridge blotting using nCounter Pro Prep, data acquisition, and data analysis.