Chapter 2 38 Evaluation of development The developmental stages for both ctns‒/‒ and wt zebrafish were monitored over the first three days of maturation at predetermined time points (3h, 6h, 24h, 48h and 72h post fertilization) according to the staging system by Kimmel et al,. 199561. The outcomes of four independent crossings involving 16 females and eight males from each genotype were used (363 ctns‒/‒ and 322 wt embryos). Percentages of different developmental stages were calculated per total number of living embryos for each genotype at each time point and compared by Chi-square test. Evaluation of apoptosis Acridine orange (AO):Morphologically sound 5dpf zebrafish wt andctns‒/‒ larvae were washed with egg water three times and immersed in5µg/ml of the fluorescent DNA binding dye AO (Sigma) for 1 hour. Cystinotic larvae were either untreated or treated with 0.1 mM of cysteamine starting at 2 hpf (N= 10 for each group). After incubation larvae were washed 3 times for 5 min each in egg water to remove residual dye. Larvae were anesthetized with 80 µg/ml tricaine methanesulfonate, then fluorescent images were obtained with the Zeiss inverted fluorescence microscope (Discovery.V8) using the AxioVision release 4.7.2 software (Zeiss, Jena, Germany). Three high magnification images were obtained for each larva (head, trunk and tail), and abnormal fluorescent spots corresponding to apoptotic areas were delineated manually and quantified using the ImageJ software (http://imagej.nih.gov/ij/). Caspase-3 immunohistochemistry: Five dpf ctns‒/‒ larvae were fixed in 4% PF, transferred to 70% ethanol and embedded in paraffin. Sections were deparaffinised, and antigen retrieval was performed. Sections were incubated with an anti–cleaved caspase-3 (Asp175) rabbit antibody (1:300; Cell Signaling, Danvers, MA, USA) overnight at room temperature. Binding of the primary antibody was visualized with labelled anti-rabbit envision antibody (DAKO, Glostrup, Denmark) and diaminobenzidine as a chromogen. Caspase-3/7 enzyme assay: Five dpf wt and ctns‒/‒ larvae were homogenized in RIPA buffer with aprotinin, sodium orthovanadate and PMSF. On average 20 larvae were homogenized per 300µl of the buffer and 3 replicates were performed for each genotype. After centrifugation supernatants were preserved at –20°C till day of analysis. Caspase-3/7 enzyme activity were assayed in 1/100 dilution of each homogenate in duplicate using a commercial luciferase based assay (Promega, Madison, WI, USA) according to manufacturer’s protocol. Enzyme activities were expressed in luminescence units (RLU)/ µg protein of each sample.
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