Monoclonal primary anti brdu antibody

To label proliferating cells, larvae were incubated in egg water containing 10 mM BrdU (Sigma) solution for 24 h or 48 h at 28.5°C starting at 1 h after neomycin treatment. Larvae were then fixed with 4% PFA overnight at 4°C. BrdU incorporation was detected by immunocytochemistry. The fixed larvae were washed three times in PBT-2 and placed in 2 N HCl for 0.5 h at 37°C. Larvae were blocked in 10% normal goat serum for 1 h at room temperature and incubated with the monoclonal primary anti-BrdU antibody (1:200 dilution; Santa Cruz Biotechnology, Inc., CA, USA) overnight at 4°C. The next day, the larvae were washed three times for 10 min each with PBT-2 and then incubated with the secondary antibody for 1 h at 37°C. Specimens were examined by Leica confocal fluorescence microscope (TCS SP5; Leica, Wetzlar, Germany).

Proliferating cells in the lateral line neuromasts were labelled by adding 10 mM 5-bromo-2-deoxyuridine (BrdU; Sigma-Aldrich) to the fresh egg water for 24 h or 48 h at 28.5°C. Larvae were then fixed with 4% PFA overnight at 4°C, and BrdU incorporation was detected by fluorescent immunostaining. The fixed larvae were washed three times in PBS containing 0.5% Triton X-100 (PBT-2) and placed in 2 N HCl for 0.5 h at 37°C. Larvae were blocked in 10% normal goat serum for 1 h at room temperature and incubated with the monoclonal primary anti-BrdU antibody (1:200 dilution; Santa Cruz, Dallas, TX, USA. Cat. no. sc-32323) overnight at 4°C. The next day, larvae were washed three times for 10 min each with PBT-2 and then incubated with the secondary antibody for 1 h at 37°C. Fluorescently labelled larvae were imaged with a Leica confocal fluorescence microscope (TCS SP5; Leica, Wetzlar, Germany). Images were processed using Photoshop software (Adobe).