{
  "abstract": "Background The gold standard for chromogenic and fluorescent immunohistochemistry (IHC) in translational research and clinical diagnostics relies on HRP-tagged secondary antibodies. While widely adopted, these enzyme-based systems can introduce background signal, display enzymatic diffusion artifacts, and limit multiplexing capabilities. To address these limitations, we demonstrate the HCR™ HiFi Encoder—a plug-and-play reagent that functionalizes unmodified primary antibodies for direct compatibility with HCR™ Pro detection, a next-generation signal amplification system supporting both fluorescent and chromogenic detection with clinical-grade sensitivity. The HCR™ HiFi Encoder incorporates MI’s patented background suppression technologies, enabling high-performance protein imaging.Methods The HCR™ HiFi Encoder binds specifically and with high affinity to the Fc-domain of native primary antibodies, forming a stable 1:1 docking complex without altering antigen-binding affinity. The method is compatible with antibodies in their native buffer formulations and does not require purification or chemical modification. It can be used with as little as 0.5 µg of primary antibody, preserving valuable or limited antibody stocks. To ensure detection methods were deployed across a range of tissue types, fixation times, and clinically relevant antigens, encoded primary antibodies were applied to a combination of commercially available and clinically derived FFPE human tonsil, lung autopsy, and breast biopsy samples followed by detection with HCR™ Pro. Performance was benchmarked against traditional HRP-based detection methods.Results HCR™ Pro detection of HiFi-encoded antibodies yielded crisp, high-resolution staining with low background and high signal-to-noise ratios. The integration of proprietary background suppression chemistry enabled clean signal even in challenging tissue contexts. Both fluorescent and chromogenic modalities showed strong performance across varied fixation and tissue conditions, with no evidence of enzymatic diffusion or tissue damage. The superior sensitivity of HCR™ Pro enabled clear detection of low-abundance targets. Notably, HCR™ Pro supports up to 2-plex detection without the need for harsh chemical stripping steps typically required for HRP-based multiplexed IHC.Conclusions The HCR™ HiFi Encoder enables high-sensitivity detection of unmodified primary antibodies using HCR™ Pro. This 1:1 docking system preserves antibody functionality, incorporates proprietary background suppression chemistry, and requires only minimal antibody input—supporting conservation of rare or expensive reagents. With flexible output modes (fluorescence or chromogenic) and parallel 2-plex detection without harsh stripping, the HCR™ HiFi Encoder + HCR™ Pro system offers a powerful and practical alternative to HRP-based detection methods, with strong translational potential for clinical diagnostics.",
  "authors": [
    {
      "affiliations": [
        "National Emerging Infectious Diseases Laboratories, Boston University, Boston, MA, USA"
      ],
      "name": "Nick Crossland"
    },
    {
      "affiliations": [
        "Molecular Instruments, Los Angeles, CA, USA"
      ],
      "name": "Chun Hao Randy Chen"
    },
    {
      "affiliations": [
        "Molecular Instruments, Los Angeles, CA, USA"
      ],
      "name": "Cameron Earl"
    },
    {
      "affiliations": [
        "Molecular Instruments, Los Angeles, CA, USA"
      ],
      "name": "Aneesh Acharya"
    },
    {
      "affiliations": [
        "Molecular Instruments, Los Angeles, CA, USA"
      ],
      "name": "Harry Choi"
    }
  ],
  "title": "70 HCR™ HiFi Encoders enable straightforward detection of unmodified primary antibodies in IHC with HCR™ Pro",
  "uid": "24eedd72-abac-5321-9634-e038cdf396cf"
}