Alexa fluor 640

For immunofluorescence staining, the embryos were anesthetized and then fixed using 4% paraformaldehyde. After washing three times with PBS-T, the embryos were incubated in the antigen retrieval solution (Beyotime Biotechnology, China, #P0088) for 15 min at 98°C. Non-specific binding was then blocked with 10% donkey serum (Solarbio, China, #SL050) in PBS-T. Next, specific primary antibodies against GFP (Abcam, #ab13970) and cleaved caspase-3 (CST, #9664), 5-bromo-2′-deoxyuridine (BrdU) (Sigma, #B5002) or SOX2 (Abcam, #ab97959) were added, and secondary antibodies were used to detect the primary antibodies.
TdT-mediated dUTP nick end labeling (TUNEL) assay was performed according to the manufacturer’s instructions (Alexa Fluor 640, cat#: 40308ES20, YEASEN Biotech Co. Ltd) to detect cell death in the HCs of neuromast. In brief, the embryos were anesthetized and then fixed using 4% paraformaldehyde. After washing three times with PBST, 20 μg/mL proteinase K (Roche) was used to treat the embryos. Next, Alexa Fluor 640-12-dUTP Labeling Mix was applied to label the apoptotic cells for at least 3 h. DAPI was applied to label the nucleus.
Images were taken with a Nikon confocal microscope A1R at 40× magnification and were analyzed by Nikon A1R NIS Elements. Exposure settings were adjusted to minimize oversaturation.

For the cell apoptosis, we used a TUNEL assay. In brief, the PFA-fixed 7 dpf zebrafish larvae in each group were permeabilized using 10 μg/ml proteinase K for 30 min, and the apoptotic cells were stained using a TUNEL apoptosis detection kit Alexa Fluor 640 (YEASEN, 40308ES20) according to the manufacturer’s instructions. Images were captured by Leica SP8 microscope.