1. Oligonucleotide conjugated antibody strategies for cyclic immunostaining.
- Author
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Jones, Jocelyn A., McMahon, Nathan P., Zheng, Ting, Eng, Jennifer, Chin, Koei, Kwon, Sunjong, Nederlof, Michel A., Gray, Joe W., and Gibbs, Summer L.
- Subjects
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IMMUNOSTAINING , *IMMUNOGLOBULINS , *OLIGONUCLEOTIDES , *IMMUNOFLUORESCENCE , *PROTEOMICS , *FLUORESCENCE - Abstract
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. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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