Abstract 2098: Development and testing of specimen-protecting microscopy consumables that enable the automation of cyclic immunofluorescence methodology

Abstract Introduction: Spatial biology provides unparalleled proteomic and transcriptomic information on the organization of the tumor microenvironment. However, the manual handling and processing of microscope slides hinder the clinical impact of cutting-edge techniques. Experiment and Data: We introduce and test “image-ready” coverpads to automate sample preparation workflows for multiplex immunofluorescence and immunohistochemistry methods on the Parhelia Spatial Station™ system to acquire single cell level spatial information on whole sides. These “permanent” coverpads with 0.07 mm spacers and thickness No. 1.5 simultaneously provide a reaction chamber for capillary-gap application of staining and washing reagents while protecting specimens from damage generally occurring during manual handling, and allow for imaging without the need for coverslip exchange. Image-ready coverpads were applied to different cyclic immunofluorescence (CycIF) assays using automation on the robot, whereby reagents (multiple antibodies or reporters) are cyclically applied, imaged, stripped, and re-applied to build a larger multiplex target panel. The final step in each cycle of these methods is to immerse the specimen in mounting medium and apply coverslip for imaging. Image-ready coverpads combined with Parhelia’s mounting medium that is fluid enough to be applied by the Spatial Station, allowed multi-cycle imaging on multiple microscopes/scanners (Zeiss Axioscanner, Phenocycler Fusion using adapted settings) without fading and tissue damage, and also saving significant amount of time by not having to remove coverslips. These experiments demonstrate the coverpad utility in efficient and consistent antibody staining in cyclic methods. Ongoing studies with assay manufacturers and academic collaborators will further extend and optimize automated protocols to other assays, expanding the utility of these coverpads to a menu of technologies. Conclusion: Overall, Image Ready coverpads provide a facile mechanism enabling the standardization of cyclic immunofluorescence and immunohistochemistry techniques, an essential milestone in translating spatial biology from research to clinical settings. Citation Format: Michael Craft, Noemi Kedei, Nels Wedin, Geoffrey K. Feld, Nikolay Samusik. Development and testing of specimen-protecting microscopy consumables that enable the automation of cyclic immunofluorescence methodology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2098.