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

Anugraha Rajagopalan, Ishwarya Venkatesh, Rabail Aslam, David Kirchenbuechler, Shreyaa Khanna, David Cimbaluk, Jeffrey H. Kordower, Vineet Gupta

Research output: Contribution to journalArticlepeer-review

9 Scopus citations


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 languageEnglish (US)
Article number100006
JournalCell Reports Methods
Issue number2
StatePublished - Jun 21 2021
Externally publishedYes


  • 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


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