(a?d) Acridine orange: Five dpf wt larvae and ctns?/? larvae, naïve to treatment or treated with 0.1?mM of cysteamine (N?=?10 for each group), were incubated with Acridine Orange (AO). Fluorescent spots (white arrows) were delineated in high magnification mode and quantified by ImageJ software. (a) A representative tail segment of 5 dpf wt larva (bar?=?200??m). (b) A representative tail segment of 5 dpf ctns?/? untreated larva (bar?=?200??m). (c) A representative tail segment of 5 dpf ctns?/? larva treated with 0.1?mM cysteamine (bar?=?200??m). (d) Quantitation of the relative fluorescence intensity of apoptotic spots. Average intensity of untreated ctns?/? larvae was set at 100%. *** P?<?0.001 against untreated ctns?/? larvae. (e,f) Caspase-3 immunohistochemistry. (e) Representative images showing increased apoptotic signal over the proximal tubule in 5 dpf ctns?/? larva (left) compared to the negative control (right), bar?=?10??m. pt, proximal tubule. (f) Representative images showing increased apoptotic signal over the liver in 5?dpf ctns?/? larva (left) compared to the negative control (right), bar?=?30??m. Rabbit serum was used for the negative control sections instead of 1ry Ab. (g) Caspase-3/7 enzyme activity. Quantitation of Caspase-3/7 enzyme activity by a luciferase based assay in the homogenates of 5 dpf wt and ctns?/? larvae (On average 60 larvae over 3 separate homogenates for each genotype were used). Results were expressed in luminescence units (RLU)/?g protein of each homogenate. ***P?<?0.001.
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