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Signal removal methods for highly multiplexed immunofluorescent staining using antibody conjugated oligonucleotides
Conference proceeding   Open access

Signal removal methods for highly multiplexed immunofluorescent staining using antibody conjugated oligonucleotides

Nathan McMahon, Jocelyn Jones, Jennifer Eng, Sunjong Kwon, Young-Hwan Chang, Guillume Thibault, Koei Chin, Michel Nederlof, Joe Gray and Summer L. Gibbs
IMAGING, MANIPULATION, AND ANALYSIS OF BIOMOLECULES, CELLS, AND TISSUES XVII, v 10881, pp 108810X-108810X-7
01 Feb 2019
PMID: 32280155
url
https://europepmc.org/articles/pmc7145771?pdf=renderView
Accepted (AM)Open Access (License Unspecified) Open

Abstract

Engineering Engineering, Biomedical Life Sciences & Biomedicine Medical Laboratory Technology Optics Physical Sciences Science & Technology Technology
Successful cancer treatment continues to elude modern medicine and its arsenal of therapeutic strategies. Therapy resistance is driven by significant tumor heterogeneity, complex interactions between malignant, microenvironmental and immune cells and cross talk between signaling pathways. Advances in molecular characterization technologies such as next generation sequencing have helped unravel this network of interactions and have vastly affected how cancer is diagnosed and treated. However, the translation of complex genomic analyses to pathological diagnosis remains challenging using conventional immunofluorescence (IF) staining, which is typically limited to 2-5 antigens. Numerous strategies to increase distinct antigen detection on a single sample have been investigated, but all have deleterious effects on the tissue limiting the maximum number of biomarkers that can be imaged on a single sample and none can be seamlessly integrated into routine clinical workflows. To facilitate ready integration into clinical histopathology, we have developed a novel cyclic IF (cycIF) technology based on antibody conjugated oligonucleotides (Ab-oligos). In situ hybridization of complementary oligonucleotides (oligos) facilitates biomarker labeling for imaging on any conventional fluorescent microscope. We have validated a variety of oligo configurations and their respective signal removal strategies capable of diminishing fluorescent signal to levels of autofluorescence before subsequent staining cycles. Robust signal removal is performed without the employment of harsh conditions or reagents, maintaining tissue integrity and antigenicity for higher dimensionality immunostaining of a single sample. Our platform Ab-oligo cycIF technology uses conventional fluorophores and microscopes, allowing for dissemination to a broad audience and congruent integration into clinical histopathology workflows.

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Web of Science research areas
Engineering, Biomedical
Medical Laboratory Technology
Optics
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