Multiplex immunofluorescence enabled by robust antibody encoding with HCR™ HiFi Encoder

Molecular Instruments, announces the forthcoming release of the HCR™ HiFi Encoder, a new strategy for multiplex immunofluorescence (IF) that integrates quantitative signal amplification directly into user-supplied primary antibodies.

Traditional multiplex IF approaches are limited by two longstanding constraints:

• Dependence on fixed panels of validated primary antibodies that often restrict experimental flexibility.
• The use of secondary antibodies, which prevents same-species/isotype combinations within a single experiment.

The HCR™ HiFi Encoder addresses both challenges by enabling direct encoding of amplification handles onto user-selected primaries, without affecting antigen-binding affinity. This permits multiplexed IF with multiple primary antibodies from the same host species and isotype, eliminating the conventional tradeoffs that have shaped IF practice for decades.

The approach builds on advances from HCR™ Gold, a quantitative 1-step amplification method that employs small, non-interacting DNA hairpins to penetrate tissues and undergo triggered polymerization at target sites. This process yields high signal-to-background ratios and deep tissue penetration (up to 1 cm), extending to both protein and RNA targets. With the HiFi Encoder, encoded primary antibodies can be used for HCR™ Gold IF and combined seamlessly with HCR™ Gold RNA-FISH, enabling simultaneous 10-plex protein and RNA imaging within a single specimen.

Seasoned IF practitioners are all too aware that existing IF products face an unfortunate tradeoff – either limiting flexibility through imposition of predetermined panels for technologies that are not robust when applied to user’s primaries of choice, or else limiting multiplexing through use of secondary antibodies that prohibit use of same-species/isotype primaries. The HCR™ HiFi Encoder escapes this tradeoff by giving researchers the flexibility to robustly and routinely encode best-in-class signal amplification to their trusted primary antibodies. We are excited for the HCR™ HiFi Encoder and HCR™ Gold IF to unleash the creativity of researchers that have long been impeded by the historical constraints of IF technologies.”

Dr. Aneesh Acharya, Chief Commercial Officer at Molecular Instruments.

Key features include:

• Robust encoding — Encode your favorite primaries without affecting target-binding affinity.
• Same-species multiplexing — Perform multiplex IF using multiple primary antibodies from the same host species and isotype with no cross-reactivity.
• Best-in-class amplification — Use 1-step quantitative HCR™ Gold signal amplification for protein imaging, leveraging the dynamic nanotechnology that has redefined the state-of-the-art for RNA imaging; small amplification components penetrate the sample without interaction, then execute triggered growth of bright amplification polymers at the site of targets, achieving exceptional signal-to-background ratios and unprecedented sample penetration (e.g., 1 cm).
• Unified protein and RNA imaging — Combine HCR™ Gold IF with HCR™ Gold RNA-FISH (fluorescence in situ hybridization), performing 1-step quantitative HCR™ Gold signal amplification for 10 protein and RNA targets simultaneously.

The HCR™ HiFi Encoder will be launched in 2025 as part of the HCR™ Gold platform, expanding the reach of programmable amplification technology across RNA and protein modalities.