Oligonucleotide conjugated antibody strategies for cyclic immunostaining

Jocelyn A. Jones, Nathan P. McMahon, Ting Zheng, Jennifer Eng, Koei Chin, Sunjong Kwon, Michel A. Nederlof, Joe W. Gray, Summer L. Gibbs

Research output: Contribution to journalArticlepeer-review

5 Scopus citations


A number of highly multiplexed immunostaining and imaging methods have advanced spatial proteomics of cancer for improved treatment strategies. While a variety of methods have been developed, the most widely used methods are limited by harmful signal removal techniques, difficulties with reagent production and antigen sensitivity. Multiplexed immunostaining employing oligonucleotide (oligos)-barcoded antibodies is an alternative approach that is growing in popularity. However, challenges remain in consistent conjugation of oligos to antibodies with maintained antigenicity as well as non-destructive, robust and cost-effective signal removal methods. Herein, a variety of oligo conjugation and signal removal methods were evaluated in the development of a robust oligo conjugated antibody cyclic immunofluorescence (Ab-oligo cyCIF) methodology. Both non- and site-specific conjugation strategies were assessed to label antibodies, where site-specific conjugation resulted in higher retained binding affinity and antigen-specific staining. A variety of fluorescence signal removal methods were also evaluated, where incorporation of a photocleavable link (PCL) resulted in full fluorescence signal removal with minimal tissue disruption. In summary, this work resulted in an optimized Ab-oligo cyCIF platform capable of generating high dimensional images to characterize the spatial proteomics of the hallmarks of cancer.

Original languageEnglish (US)
Article number23844
JournalScientific Reports
Issue number1
StatePublished - Dec 2021

ASJC Scopus subject areas

  • General


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