Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) is an essential tool for detecting cell death in tissues, but its compatibility with spatial proteomic methods is unknown. We evaluated variations of the TUNEL protocol for compatibility with multiple iterative labeling by antibody neodeposition (MILAN) in 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 the 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 enhanced protein antigenicity for the targets tested. Antibody-based TUNEL with pressure cooker retrieval could be flexibly integrated into a MILAN staining series, and first-round TUNEL was also compatible with a second spatial proteomic method, cyclic immunofluorescence (CycIF). We anticipate that this harmonization of TUNEL with spatial proteomics will enhance the spatial contextualization of cell death in complex tissues.
Keywords: CP: Imaging; CycIF; MILAN; TUNEL; acetaminophen liver toxicity; apoptosis; cell death; dexamethasone-induced adrenal insufficiency; proteinase K; spatial proteomics; zonation.
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