Flexible Cyclic Immunofluorescence (cyCIF) Using Oligonucleotide Barcoded Antibodies

Nathan P. McMahon, Jocelyn A. Jones, Ashley N. Anderson, Matthew S. Dietz, Melissa H. Wong, Summer L. Gibbs

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Advances in our understanding of the complex, multifaceted interactions between tumor epithelia, immune infiltrate, and tumor microenvironmental cells have been driven by highly multiplexed imaging technologies. These techniques are capable of labeling many more biomarkers than conventional immunostaining methods. However, multiplexed imaging techniques suffer from low detection sensitivity, cell loss—particularly in fragile samples—, and challenges with antibody labeling. Herein, we developed and optimized an oligonucleotide antibody barcoding strategy for cyclic immunofluorescence (cyCIF) that can be amplified to increase the detection efficiency of low-abundance antigens. Stained fluorescence signals can be readily removed using ultraviolet light treatment, preserving tissue and fragile cell sample integrity. We also extended the oligonucleotide barcoding strategy to secondary antibodies to enable the inclusion of difficult-to-label primary antibodies in a cyCIF panel. Using both the amplification oligonucleotides to label DNA barcoded antibodies and in situ hybridization of multiple fluorescently labeled oligonucleotides resulted in signal amplification and increased signal-to-background ratios. This procedure was optimized through the examination of staining parameters including staining oligonucleotide concentration, staining temperature, and oligonucleotide sequence design, resulting in a robust amplification technique. As a proof-of-concept, we demonstrate the flexibility of our cyCIF strategy by simultaneously imaging with the original oligonucleotide conjugated antibody (Ab-oligo) cyCIF strategy, the novel Ab-oligo cyCIF amplification strategy, as well as direct and indirect immunofluorescence to generate highly multiplexed images.

Original languageEnglish (US)
Article number827
JournalCancers
Volume15
Issue number3
DOIs
StatePublished - Feb 2023

Keywords

  • DNA barcoded antibodies
  • cyclic immunofluorescence (cyCIF)
  • photocleavable linkers
  • spatial proteomics

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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