Abstract
AbstractTerminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) is an essential tool for the detection of cell death in tissues. Although TUNEL is not known to be compatible with multiplexed spatial proteomic methods, harmonizing TUNEL with such methods offers the opportunity to delineate cell-type specific cell death labeling and precise spatial contextualization of cell death in complex tissues. Here we evaluated variations of the TUNEL assay for their compatibility with a multiplexed immunofluorescence method, multiple iterative labeling by antibody neodeposition (MILAN), in two different tissues and injury models for cell death, acetaminophen-induced hepatocyte necrosis and dexamethasone-induced adrenocortical apoptosis. Using a commercial Click-iT-based assay as a standard, TUNEL signal could be reliably produced independent of antigen-retrieval method, with tissue-specific minor differences in signal-to-noise. In contrast, proteinase K treatment consistently reduced or even abrogated protein antigenicity, while pressure cooker treatment consistently enhanced protein antigenicity for the targets tested. Antibody-based TUNEL protocols using pressure-cooker antigen retrieval were MILAN erasure-compatible thus enabling harmonization of TUNEL with MILAN. As many as four staining cycles could be performed without loss of subsequent TUNEL signal, while first-round TUNEL did not influence protein antigenicity in subsequent rounds. We conclude this harmonized assay performs comparably to an established commercial assay, but preserves protein antigenicity, thus enabling versatile integration with multiplexed immunofluorescence using MILAN. We anticipate this harmonized protocol will enable broad and flexible integration of TUNEL into multiplexed spatial proteomic assays, thus vastly enhancing the spatial contextualization of cell death in complex tissues.
Publisher
Cold Spring Harbor Laboratory