Journal: Cell reports methods

Article Title: Harmonizing TUNEL with multiplexed iterative immunofluorescence enriches spatial contextualization of cell death.

doi: 10.1016/j.crmeth.2025.101047

Figure Lengend Snippet: Figure 2. PC-IH preserves TUNEL signal in apoptosis (A) Mice were treated with DEX, DEX+COS, or DMSO, and the adrenal glands were harvested at 4 days for Click-iT (top row) or PC-IH (bottom row) protocol TUNEL staining. (B and C) Quantification of TUNEL+ nuclei over total cortical (zF + zG) nuclei by Click-iT (B) or pressure cooker (C). (D) Signal to noise for both protocols, where TUNEL+ signal is divided by TUNEL− DAPI spots. Error bars are SEM; significance is defined by t test, *p < .05; **p < .01. Scale bars: 200 μm. See also Figure S2.

Article Snippet: The first is consistent with prior studies showing that cleaved caspase-3 activation occurs early in apoptosis2,47 and is even reversible in some cell types,48,49 whereas TUNEL labels DNA fragmentation, which is a later, terminal event.50–52 Nuclear remnantsmay retain TUNEL positivity evenafter cytoplasmic components, including cleaved caspase-3, have degraded.50,51,53 The second phenomenon—TUNEL signal occurring in DAPI-poor regions—is similarly observed in published wide-field images, where apoptotic DNA fragmentation often coincides with a reduced or absent DAPI signal.54,55 A review of exemplar images from major TUNEL kit manufacturers further demonstrates TUNEL positivity on a backdrop of a low- or no-DAPI signal (e.g., Cell Signaling Technology, TUNEL Assay Kit, cat. #25879; Invitrogen APOBrdU TUNEL Assay Kit, cat. #A23210), and the same pattern was reported inanother model of adrenal apoptosis.56 In our study, both markers and their unexpected colocalization patterns exhibited robust signals in DEX-treated samples with the expected fragmented morphology in the tissue context where apoptosis was first described,22 reinforcing their validity in identifying apoptotic cells.

Techniques: TUNEL Assay, Staining