Fig. 3

Quantitative analysis of apoptotic cell death by TUNEL assay.

TUNEL staining as a quantitative assay was performed to determine the protective effect of sodium selenite against neomycin-induced apoptosis. Apoptotic cells are marked as light red dots in red-colored fish after TUNEL staining as observed under a fluorescent microscope (arrow indicates TUNEL-positive cells in TUNEL reaction (b) column and merge (d) column). Light red dots were not visible in the negative control zebrafish (A). A comparison of the color intensity between the group treated with 125 μM neomycin and the group treated with 500 μM sodium selenite for 1 h showed that the number of TUNEL-positive cells significantly decreased in the 5 and 6-dpf transgenic zebrafish treated with sodium selenite, and that apoptotic cell death of the neuromasts hair cells was attenuated by sodium selenite (green and red-colored fish in the left column, combination of control and TUNEL reaction (a), × 10; red-colored fish in the left middle column, TUNEL reaction (b), × 10; green-colored fish in the right middle column, control for TUNEL reaction (c), × 10; green and red-colored neuromast in the right column, merge for control and TUNEL reaction by high magnification (d), × 40) (A). The concentration of 500 μM of sodium selenite reduced significantly neomycin-induced TUNEL positive cells in four neuromasts (SO1, SO2, O1, and OC1) (*: statistically significant) (B). Negative control: TUNEL positive cells = 0.00 ± 0.00; NM 125 μM: the average TUNEL positive cells, 4.98 ± 1.72; S 500 μM + NM 125 μM: the average TUNEL positive cells = 0.95 ± 1.47; n = 10 fish per treatment, *: statistically significant. (B). Scale bars = 200 μm (c); 20 μm (d). NM, neomycin; S, sodium selenite.