Introduction: Addressing the Limitations of Multiplex Imaging
Advances in spatial proteomics have underscored the need for highly multiplexed imaging platforms that can preserve tissue integrity while detecting both abundant and rare proteins. Conventional cyclic immunofluorescence (cyCIF) techniques struggle with low sensitivity, signal removal harshness, and limited flexibility in antibody labeling. McMahon et al. address these issues by presenting an improved cyCIF platform based on oligonucleotide-barcoded antibody conjugates (Ab-oligos), enabling sensitive, reproducible, and modular staining for complex biological tissues.
Core Innovation: The Antibody-Oligonucleotide (Ab-Oligo) Conjugate
At the heart of this platform is the Ab-oligo, an antibody covalently linked to a unique single-stranded DNA sequence known as a docking strand (DS). These DS sequences serve as programmable anchors for fluorescently labeled imaging strands (IS) that hybridize to the DS for detection. Importantly, each IS carries fluorophores linked via photocleavable linkers (PCLs), allowing for rapid, UV-based signal removal between imaging cycles—a gentler alternative to chemical stripping or bleaching, particularly beneficial for fragile samples like blood smears.
This DNA-mediated architecture introduces modularity: fluorophores are no longer permanently tethered to antibodies, and multiple oligo-based detection strategies can be adapted on a per-marker basis.
Amplifying the Signal: Solving Low-Abundance Detection
To address the challenge of detecting low-abundance antigens, the authors developed an Ab-oligo amplification strategy. Here, a long amplification strand (AmpS) hybridizes partially with the DS, leaving an overhang that can bind up to ten short fluorophore-labeled amplification imaging strands (Amp IS). This configuration boosts signal intensity dramatically—up to 10-fold compared to conventional direct labeling without compromising tissue morphology or requiring enzymatic amplification steps.
Additionally, the design of DS and AmpS sequences was systematically optimized to avoid secondary structures, ensuring robust and uniform hybridization across multiple antibody clones and targets.
Extending the Platform: Oligo-Conjugated Secondary Antibodies
Some primary antibodies do not tolerate direct chemical conjugation with oligonucleotides. To broaden applicability, the authors introduced oligo-conjugated secondary antibodies, enabling indirect labeling using standard primaries while retaining UV-cleavable imaging capability. This innovation ensures that even sensitive antibodies can be integrated into the cyCIF workflow without loss of signal specificity or intensity.
Validation and Application
The platform was validated on frozen and formalin-fixed tissues, including xenograft tumors and blood-derived samples. Using a combined strategy of direct IF, Ab-oligo, amplified Ab-oligo, and indirect oligo-based labeling, the authors generated 12-marker multiplexed images from a single tissue section. Importantly, the use of UV-cleavable IS minimized sample degradation over multiple imaging cycles—a key advantage over conventional bleaching-based approaches.
Conclusion
McMahon et al. have delivered a flexible, modular, and high-sensitivity cyCIF platform centered on the use of Ab-oligo conjugates. This system addresses the core limitations of multiplexed imaging by decoupling fluorophore detection from antibody binding, enabling signal amplification, and allowing tailored approaches per antigen. The result is a robust and adaptable method well-suited for unraveling spatial biology across diverse tissue types and conditions.