A1301

The zebrafish were fixed in ethanol: glacial acetic acid at a 3:1 ratio at 4 °C for 72 hours, then washed in 100% ethanol and rehydrated progressively in ethanol/saline solutions before staining in 0.01% acridine orange (A1301, Thermo Fisher Scientific) in phosphate-buffered saline with gentle agitation before dehydration in an ethanol/ saline series. The dehydrated specimens were washed three times in absolute ethanol (dried with molecular sieve) and then transferred via xylene, changed twice and left in xylene for two hours and checked for transparency. They were tumbled gently overnight in a solution Fluoromount^{31 (link)} (Fluoromount is no longer available commercially: we would advise Histomount (Thermo Fisher Scientific) as similar substitute) before mounting in a single-cavity slide under a standard coverslip, with the specimen left uncovered to facilitate the evaporation of the xylene solvent and more mountant being added to reduce shrinkage. Imaging was performed with glycerol immersion on the Mesolens and custom built acquisition software based on WinFluor³² .

Autophagy is characterized by the formation and promotion of acidic vesicular organelles (AVOs). In acridine orange-stained cells, the cytoplasm and nucleus exhibit bright green and dim red fluorescence, whereas acidic compartments exhibit bright red or orange fluorescence, as described previously [18 (link)]. Following drug treatment, 5 µg/mL acridine orange (A1301; Invitrogen, Carlsbad, CA, USA) was added to the culture medium, and the cells were incubated at 37 °C for 15–30 min. The cells were then trypsinized, washed twice with cold PBS, and observed under a confocal microscope. Fluorescence imaging was performed using a blue bandpass filter with 490 nm excitation, and the fluorescence of the green and orange channels was recorded and merged.

Juvenile squid were collected at 14 h post colonization, anesthetized in 2% ethanol, and placed in 0.0001% acridine orange (Invitrogen, A1301) in seawater for 1 min, as previously described [52 (link)]. Light-organ appendages were visualized to determine the number of acridine orange–positive apoptotic nuclei using fluorescence confocal microscopy.

The CBMN assay was conducted according to a previously published procedure (Fenech and Morley 1985b (link)). Briefly, cytochalasin B (5474, Tocris Bioscience, Bristol, UK, 6 μg/ml) was applied to the 3D EpiIntestinal™ in vitro tissues every other day, to the apical and to the basolateral compartments. Tissues were dissociated into single cell suspension (described above), processed with mild hypotonic cold 75 mM KCl, followed by 3 fixation steps in Carnoy’s fixative (methanol/acetic acid, 16:1), supplemented with formaldehyde at 1st fixation. The cell suspension was stored in fixative at 4 °C overnight, stained with acridine orange (1:100 dilution in 1 × PBS, A1301, Invitrogen; Thermo Fisher Scientific, Waltham, MA) and scored under fluorescence microscopy (Zeiss Axioimager, equipped with triple band excitation fluorescence filter set: DAPI, FITC and Texas red). Scoring was performed following the criteria developed by Fenech et al. (Fenech et al. 2003 (link), 2007 (link)). One thousand total cells per microtissue were scored to determine the percentage of cells with one (mononucleated) or two nuclei (binucleated). The results were generated from three independent experiments. Each experiment consisted of a single microtissue per dose.