A1r nis elements

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.

Isolation and basal podocyte [Ca²⁺]_i measurements were done as has been previously reported (Ilatovskaya and Staruschenko 2013; Ilatovskaya et al. 2015b). Briefly, the kidney cortex was mechanically isolated and minced until homogenous. The minced tissue was pushed through a 100 followed by a 140 mesh sieve. The flow‐through was filtered by a 200 mesh sieve and the sieve‐sedimented glomeruli were collected. Isolated glomeruli were incubated with Fura Red, AM and Fluo‐4, AM (5 μmol/L; Invitrogen, Grand Island, NY) for 40 min at room temperature. Glomeruli were adhered to poly‐l‐lysine coated glass coverslips and imaged using a confocal laser‐scanning microscope.
Calcium imaging was performed as has been previously described (Ilatovskaya and Staruschenko 2013; Ilatovskaya et al. 2014, 2015a,b). A laser‐scanning confocal microscope system (Nikon A1‐R, NIS Elements; Nikon Instruments Inc, Tokyo, Japan) was used to collect images in time series (xyt, 4 s per frame). Basal [Ca²⁺]_i of podocytes were measured after stable fluorescence intensities were established. Only surface podocytes of glomeruli were recorded. Each experiment measured basal [Ca²⁺]_i of 4 to 9 podocytes of at least one glomerulus imaged.