Abstract 717: Advancing spatial oncology research with open-panel multiplex IHC/IF using a novel heat-free antibody stripping reagent.

Abstract Purpose: Spatially resolved multiplex immunohistochemistry (IHC) and immunofluorescence (IF) assays are essential for profiling the tumor microenvironment and identifying biomarkers that predict response to cancer immunotherapies, yet many platforms rely on proprietary closed antibody panels and harsh, heat-based stripping that damage FFPE tissue and reduce antigenicity, limiting target choice and rapid adoption of new markers. We developed a heat-free stripping reagent that removes primary and secondary antibodies and other non-covalently bound detection reagents between staining cycles while preserving tissue architecture and antigen integrity. This abstract summarizes its performance in multiplex IHC, IF, and tyramide-based workflows and its compatibility with common autostainers. Experimental Procedures: FFPE tumor and normal tissues were stained using sequential multiplex IHC, IF and TSA workflows in both manual and automated staining, with panels targeting immune, stromal, and tumor markers. After each staining round, slides were treated with the heat-free reagent before the next cycle. Residual signal was assessed by imaging previously used channels after stripping and by chase experiments to detect previously applied antibodies. Antigen preservation was evaluated by comparing staining intensity and distribution for the same targets across early and late cycles, and morphology by H Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 717.