Multiplexed Immunofluorescence Assay for Spatial Assessment of Senescence Markers in vivo

Multiplexed immunofluorescence (IF) staining represents a step-change in sophistication over traditional IF imaging allowing sequential detection of multiple antibody markers within a single tissue section. This facilitates profiling of a wider range of cell types and tissue structures, as well as allowing assessment of the spatial relationship between these in a way that is not feasible with sequential sections. There is no single universal marker of senescence, and it is now widely appreciated that senescence signatures vary by cell type, senescence trigger and time since induction. Thus, the combinatorial approaches offered by multiplex IF provide a powerful tool to enable unbiased senescent cell detection within a single tissue section. Here, tissue microarrays containing cores from 34 human tissue types were employed. These were stained with a combination of structural, immune and senescence biomarkers in a 10-plex assay, with images acquired using a multiplex slide scanner. This method allows assessment of the abundance and spatial distribution of senescent cells in tissue. Sequential staining, imaging, and dye inactivation cycles were used to generate multiplex datasets, with images acquired using a multiplex fluorescence slide scanning system. Protocols for both manual slide preparation and automated processing using a slide stainer are described, together with single-cell segmentation and spatial analysis using digital pathology software. Overall, these methods provide a comprehensive guide for the staining, imaging and spatial analysis of senescence-associated biomarkers within tissue samples as well as a more general guide to multiplexed imaging.