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Fig. 4 Measurement of apoptosis using acridine orange (AO) staining and intracellular reactive oxygen species (ROS) using DCFH-DA staining in PTZ-induced epilepsy-like conditions in zebrafish larvae. (Scale bar: 100 μm). (A) AO-stained larvae, (B) DCFH-DA-stained larvae, (C) quantification of AO fluorescence intensity, and (D) quantification of DCFH-DA fluorescence intensity. Fluorescent intensities were analyzed using ImageJ software. Experimental groups (n = 15/group) included zebrafish larvae treated with N3 at 50 µM, N3 at 100 µM, PTZ (6 mM), positive control (PC) Valproic acid at 100 µM, and control group. Each assay was conducted in triplicate with three independent experiments. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. Significant differences between tested samples and the negative control are indicated by ****p < 0.0001