*2.7. Whole-Mount TUNEL Assay*

Whole-mount TUNEL staining was performed to evaluate apoptosis in zebrafish at 72 hpf. We washed the zebrafish with deionized water three times after exposure to Ag/ZnONPs. Zebrafish larvae were fixed with 4% paraformaldehyde for 1 h and then washed with PBS three times. Next, the embryos were incubated with blocking buffer on a shaker for 30 min and subsequently rinsed with PBS three times for 5 min. Then, the embryos were incubated with a permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate) for 30 min on ice and washed with PBS three times on a shaker for 5 min each. Finally, the TUNEL reaction mixture (labeling solution:enzyme solution = 9:1) was incubated with embryos in a water bath at 37 ◦C for 1 h, and we observed the embryos using a confocal fluorescence microscope (Nikon TE2000EPFS-C1-Si, Tokyo, Japan).
