E added to the sonicated solution. The control solution was exactly

E added to the sonicated solution. The control solution was exactly the same except without ezetimibe or hawthorn extract. After incubation in food solution for 2 hours, fish were extracted from the treatments and allowed to swim in tank water overnight. The next morning fish were imaged as described below.Materials and MethodsInitially in this section parameters and techniques common to all experiments are discussed including zebrafish husbandry (1a) and the preparation of MHE (1b). We then describe the feeding regimen (2a) and data acquisition process (2b) for the automated inhibitor hypercholesterolemia screen. Then a second set of experiments, performed with a different methodology than the hypercholesterolemia screen, are described for automated measurement of MHE’s influence on cardiodynamics. For these experiments the feeding regimen (3a), data acquisition (3b), and computational algorithms (3c and 3d) employed in assessing cardiodynamic data are described. Finally a description of statistical tests utilized is provided (4).2b. Automated Hypercholesterolemia ScreenAutomated acquisition was performed in Perkin-Elmer’s Opera high-throughput/high-content automated confocal system in 384well plates with one anesthetized fish in each well in 20 mL of tank water. The Opera system scans a user-designated area of the well in the x-y direction and focuses on a user-defined displacement on the z-axis. Nine z-stacks were obtained at different x-y locations in each well and 6 z-slices were taken per stack in a total z-range of 250 mm at a spacing of 50 mm between each z-slice. This lead to 54 total images per well (Figure 1A). The orientation of the fish in each well was random. Mean fluorescence intensity from each group of 54 images (of one individual fish) was considered a data point, except in figure 1B and 1C, where error represents the error in the mean value of the group of 54 images. In all experiments utilizing the Opera system (figures 1 and 2) ImageJ was utilized for fluorescence quantification.1a. Zebrafish HusbandryAdult zebrafish were housed in the jointUniversity of Cincinnati (UC)-Cincinnati Children’s Hospital Medical Center (CCHMC) zebrafish facility. All zebrafish husbandry and experimental procedures were performed in accordance with and approved by theUC Institutional Animal Care and Use Autophagy Committee (IACUC, protocol # 1D03020. Embryos were generated for this study from in-house lines of adult fish being bred, raised, and cared for according to established procedures [20]. Water conditions in this facility (pH = 7.1?.4; temperature = 26.5?8.5uC; conductivity = 490?30 mS; and dissolved oxygen concentration = 5.0?7.5 mg L21) were rigorously maintained through real-time computerized monitoring and dosing. For this study, transgenic TG(kdrl:mCherry) zebrafish with mCherry fluorescent protein driven by the cardiovascular specific kdrl promoter were crossed with a casper line containing a melanocyte/iridophore mutation [21]. The resulting double transgenic animals TG(Kdrl:mCherry)/Casper express red fluorescence in the vascular walls and are optically transparent through adulthood. In all data acquisition procedures fish were anesthetized in 125?50 mg L21 MS-222 (tricaine) and mounted in 1.2 agarose in glass bottomed viewing slides.3a. Feeding for Heart Beat MeasurementFive dpf, Kdrl:casper fish were fed as above except that no ezetimibe or BOD-CH were administered and 6.5 mg/mL hawthorn extract was mixed into the egg solution. Fish were.E added to the sonicated solution. The control solution was exactly the same except without ezetimibe or hawthorn extract. After incubation in food solution for 2 hours, fish were extracted from the treatments and allowed to swim in tank water overnight. The next morning fish were imaged as described below.Materials and MethodsInitially in this section parameters and techniques common to all experiments are discussed including zebrafish husbandry (1a) and the preparation of MHE (1b). We then describe the feeding regimen (2a) and data acquisition process (2b) for the automated hypercholesterolemia screen. Then a second set of experiments, performed with a different methodology than the hypercholesterolemia screen, are described for automated measurement of MHE’s influence on cardiodynamics. For these experiments the feeding regimen (3a), data acquisition (3b), and computational algorithms (3c and 3d) employed in assessing cardiodynamic data are described. Finally a description of statistical tests utilized is provided (4).2b. Automated Hypercholesterolemia ScreenAutomated acquisition was performed in Perkin-Elmer’s Opera high-throughput/high-content automated confocal system in 384well plates with one anesthetized fish in each well in 20 mL of tank water. The Opera system scans a user-designated area of the well in the x-y direction and focuses on a user-defined displacement on the z-axis. Nine z-stacks were obtained at different x-y locations in each well and 6 z-slices were taken per stack in a total z-range of 250 mm at a spacing of 50 mm between each z-slice. This lead to 54 total images per well (Figure 1A). The orientation of the fish in each well was random. Mean fluorescence intensity from each group of 54 images (of one individual fish) was considered a data point, except in figure 1B and 1C, where error represents the error in the mean value of the group of 54 images. In all experiments utilizing the Opera system (figures 1 and 2) ImageJ was utilized for fluorescence quantification.1a. Zebrafish HusbandryAdult zebrafish were housed in the jointUniversity of Cincinnati (UC)-Cincinnati Children’s Hospital Medical Center (CCHMC) zebrafish facility. All zebrafish husbandry and experimental procedures were performed in accordance with and approved by theUC Institutional Animal Care and Use Committee (IACUC, protocol # 1D03020. Embryos were generated for this study from in-house lines of adult fish being bred, raised, and cared for according to established procedures [20]. Water conditions in this facility (pH = 7.1?.4; temperature = 26.5?8.5uC; conductivity = 490?30 mS; and dissolved oxygen concentration = 5.0?7.5 mg L21) were rigorously maintained through real-time computerized monitoring and dosing. For this study, transgenic TG(kdrl:mCherry) zebrafish with mCherry fluorescent protein driven by the cardiovascular specific kdrl promoter were crossed with a casper line containing a melanocyte/iridophore mutation [21]. The resulting double transgenic animals TG(Kdrl:mCherry)/Casper express red fluorescence in the vascular walls and are optically transparent through adulthood. In all data acquisition procedures fish were anesthetized in 125?50 mg L21 MS-222 (tricaine) and mounted in 1.2 agarose in glass bottomed viewing slides.3a. Feeding for Heart Beat MeasurementFive dpf, Kdrl:casper fish were fed as above except that no ezetimibe or BOD-CH were administered and 6.5 mg/mL hawthorn extract was mixed into the egg solution. Fish were.

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