SeqStain using fluorescent-DNA conjugated antibodies allows efficient, multiplexed, spatialomic profiling of human and murine tissues

Spatial organization of molecules and cells in complex tissue microenvironments provides essential cues during healthy growth and in disease. Novel techniques are needed for elucidation of their spatial relationships and architecture. Although a few multiplex immunofluorescence based techniques have been developed for visualization of the spatial relationships of various molecules in cells and tissues, there remains a significant need for newer methods that are rapid, easy to adapt and are gentle during the cyclic steps of fluorescence staining and de-staining. Here, we describe a novel, multiplex immunofluorescence imaging method, termed SeqStain, that uses fluorescent-DNA labelled antibodies for immunofluorescence staining of cells and tissues, 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, such as Fabs, to efficiently analyse complex cells and tissues in multiple rounds of staining and de-staining. 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 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. ### Competing Interest Statement A.R. and V.G. are inventors on patent applications related to these studies. V.G. is a co-founder of 149 Bio, LLC that is licensing this intellectual property and has significant financial interest in it. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.