Chapter 2 26 Figure 4. Apoptosis in ctns‒/‒ larvae. (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= 200µm). (b) A representative tail segment of 5 dpf ctns‒/‒untreated larva (bar= 200µm). (c) A representative tail segment of 5 dpf ctns‒/‒ larva treated with 0.1 mM cysteamine (bar= 200µm). (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= 10µm. 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= 30µm. 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)/ µg protein of each homogenate. *** P<0.001.
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