Background
Multiplex immunofluorescence offers the ability to elucidate protein expression in the tumor microenvironment, but it has significant tradeoffs between sensitivity, multiplexing, and throughput. Technologies with high multiplexing capability (12-40 biomarkers) often lack robust signal amplification, resulting in lower sensitivity and sample throughput due to longer imaging times. Conversely, technologies with signal amplification often do not easily support staining larger umbers of markers, as ensuring consistent amplification across multiple markers and staining rounds is challenging. Ultivue InSituPlex® technology enables simultaneous amplification of any number of markers in one assay, which can be interrogated using multiple rounds of barcode probe hybridization with spectrally distinct dyes. In this work we demonstrate a simple workflow for highplex staining and imaging of InSituPlex® assays using the Parhelia Omni-Stainer™ and Image-Ready Coverpads. This workflow allows for rounds of probe exchange and imaging to be carried out without labor-intensive de-coverslipping steps that currently limit the number of iterative signal exchange cycles and may risk tissue damage.
Methods
FFPE tissue slides were stained using InSituPlex® assay reagents on the Parhelia Omni-Stainer™. Slides were placed underneath Image-Ready Coverpads (#2.0 glass), mounted using Parhelia Transient Mounting Medium, and scanned on a Zeiss Axioscan.Z1. Images were co-registered and analyzed using Ultivue’s STARVUE™ image data science platform, and positive cell density and staining intensity were calculated and plotted.
Results
Tissue slides stained on the Omni-Stainer™ showed robust and strong staining. While underneath Image-Ready Coverpads, the stability of Parhelia Transient Mounting Medium was demonstrated for up to 7 days without drying out and without special handling or loss of stain quality. Mounting medium was effectively removed by washes on the Omni-Stainer™ allowing for successive rounds of probe exchange and reimaging without needing to remove or reapply coverpads. Higher-plex staining was demonstrated without any need for coverslip removal or replacement throughout the imaging process, eliminating possible tissue loss due to rough de-coverslipping methods.
Conclusions
We have demonstrated a workflow for higher-plex InSituPlex® staining using the Parhelia Omni-Stainer™, combining the high sensitivity afforded by InSituPlex amplification with a newly improved workflow requiring no coverslipping or de-coverslipping between rounds of probe visualization. This opens the door to high-quality high-plex immunofluorescence assays with high sample throughput on an open and affordable stainer platform for translational research.