Streamlined high-plex imaging with InSituPlex®and ZEISS Axioscan 7 for spatial phenotyping

Multiplex immunofluorescence (mIF) provides a powerful means to visualize complex biomarker signatures with spatial context. Yet, most high-plex mIF approaches depend on repeated cycles of staining and imaging, which introduce workflow complexity and necessitate either labor-intensive manual handling or sophisticated instrumentation capable of automating these multistep procedures. Vizgen's InSituPlex assay supports rapid, multiplexed detection of up to 12 protein biomarkers in a single staining step, leveraging signal amplification to achieve robust detection of both abundant and lowly expressed targets using standard fluorescence filter configurations. When combined with the Axioscan 7 spatial biology platform, engineered for fully automated, high-throughput slide scanning with additional fluorescent channels, this workflow enables higher-plex, single-round imaging without the need for spectral unmixing. Here, we demonstrate the power of this integrated approach to streamline assay development, expand biomarker panel design flexibility, and deliver efficient, scalable multiplexed protein imaging. A next generation InSituPlex® assay was developed to increase the simultaneous detection of markers using the Axioscan 7 spatial biology system. A panel of fluorophores was carefully selected to minimize spectral overlap and maximize detection specificity, ensuring clear distinction of all fluorescent channels without requiring spectral unmixing to deconvolute signals. Signal quality was analyzed using the STARVUE image analysis pipeline which precisely determined positive cell signal intensity and density. The increased number of fluorophores showed excellent signal to noise ratio and no crosstalk between fluorescent channels. The expanded channel capacity of InSituPlex assay allows for a simplified workflow while increasing the number of biomarkers that can simultaneously be detected in tumor samples. This capability further enhances flexibility for InSituPlex panel design and streamlines the assay development process. The integration of the novel InSituPlex fluorescent dye panel and the Axioscan 7 spatial biology platform delivers a superior, high throughput mIF workflow. This combination enables higher-plex imaging while significantly reducing manual steps, leveraging the established reproducibility and throughput of both technologies. We anticipate this integrated platform will empower researchers to unlock deeper, high resolution spatial insights into cancers and their complex environments.