Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues

Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings.

IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization.