Immunofluorescence visualization of cells in the retina of 5 dpf zebrafish larvae. (a) Photoreceptors (orange arrows) were stained with anti-opsin antibodies (green), and amacrine cells (yellow arrows) were stained with GABA antibodies (red). Nuclei were stained with Hoechst 33342 (blue). (b) (left) Representative retina image stained as above. (middle) Masks covering the inner nuclear (INL) and photoreceptor layers (PL) (right). Labeled nuclei contained in those layers. (c) Cell densities among the investigated retina layers, estimated as the number of cells that were positive for opsin or GABA above the fluorescence and size thresholds and normalized to surface volume of the mask. The photoreceptor layer and INL were 1.29 × and 1.78 × larger in control retinas of 5 dpf larvae. n = 7 stim2 KO, 5 control. The scatter dot plots represent mean values ± standard error of the mean. In the case of the photoreceptor cell density analysis, the Mann–Whitney test was conducted because the data did not pass the Shapiro–Wilk normality test. Statistical analyses of INL cell density were performed using an unpaired t-test with Welch’s correction because the data passed the Shapiro–Wilk normality test. The Rout method for eliminating outliers did not reveal any outliers among the tested conditions.
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