Abstract
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.
| Original language | English (US) |
|---|---|
| Article number | 100006 |
| Journal | Cell Reports Methods |
| Volume | 1 |
| Issue number | 2 |
| DOIs | |
| State | Published - Jun 21 2021 |
Funding
We thank Liudmila Romanova and Diptaman Chatterjee in Kordower laboratory at Rush University Medical Center and the Center for Advanced Microscopy at Northwestern University for help with tissue staining and image analysis, and all past and current members of the Gupta laboratory for their technical help and suggestions. This work was supported in part by funding from the Oppenheimer Family Foundation, Bears Care, the Department of Internal Medicine at Rush University Medical Center, and by the National Institutes of Health (grants R01DK107984, R01DK084195, and R01CA244938 to V.G.). Conceptualization, V.G. A.R. and I.V.; methodology, A.R. I.V. R.A. D.K. J.H.K. and V.G.; investigation, A.R. I.V. R.A. and S.K.; software, A.R. S.K. and D.K.; supervision, V.G.; writing – original draft, A.R. I.V. R.A. D.K. S.K. D.C. and V.G.; writing – review & editing, A.R. I.V. J.K. and V.G.; funding acquisition, V.G. A.R. and V.G. are inventors on a patent application filed by Rush University Medical Center related to these studies. V.G. is a co-founder of 149 Bio, LLC and Spatialomyx, LLC that is licensing this intellectual property and has significant financial interest in it. V.G. is also an Officer and a Board member of 149 Bio, LLC and Spatialomyx, LLC. The remaining authors declare no competing interests. We thank Liudmila Romanova and Diptaman Chatterjee in Kordower laboratory at Rush University Medical Center and the Center for Advanced Microscopy at Northwestern University for help with tissue staining and image analysis, and all past and current members of the Gupta laboratory for their technical help and suggestions. This work was supported in part by funding from the Oppenheimer Family Foundation , Bears Care , the Department of Internal Medicine at Rush University Medical Center , and by the National Institutes of Health (grants R01DK107984 , R01DK084195 , and R01CA244938 to V.G.).
Keywords
- Spatialomics
- antibody DNA conjugates
- cellular neighborhoods
- multiplex immunofluorescence
- relationship maps
- sequential staining
- spatial pathology
ASJC Scopus subject areas
- Biotechnology
- Biochemistry
- Biochemistry, Genetics and Molecular Biology (miscellaneous)
- Genetics
- Radiology Nuclear Medicine and imaging
- Computer Science Applications
