SeqStain is an efficient method for multiplexed, spatialomic profiling of human and murine tissues

Abstract

Summary: Spatial organization of molecules and cells in complex tissue microenvironments provides essential organizational cues in health and disease. A significant need exists for improved visualization of these spatial relationships. Here, we describe a multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA-labeled antibodies for immunofluorescent staining and nuclease treatment for de-staining that allows selective enzymatic removal of the fluorescent signal. SeqStain can be used with primary antibodies, secondary antibodies, and antibody fragments to efficiently analyze complex cells and tissues. Additionally, incorporation of specific endonuclease restriction sites in antibody labels allows for selective removal of fluorescent signals while retaining other signals that can serve as marks for subsequent analyses. The application of SeqStain on human kidney tissue provided a spatialomic profile of the organization of >25 markers in the kidney, highlighting it as a versatile, easy-to-use, and gentle new technique for spatialomic analyses of complex microenvironments. Motivation: Spatial profiling of molecules in complex microenvironments requires techniques for multiplex measurements. Immunofluorescence-based imaging has significant advantages for such spatialomic analyses. Yet newer techniques are needed that are easy to use, utilize off-the-shelf reagents, can be applied by using readily available instrumentation, and provide data with good signal-to-noise ratio and are gentle enough to not harm the tissues.

Keywords

• Spatialomics
• multiplex immunofluorescence
• cellular neighborhoods
• spatial pathology
• relationship maps
• antibody DNA conjugates