Computational protocol: Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease

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Protocol publication

[…] Live Tg(fabp10:dsRed) larvae were treated and imaged as described (; ). Tg(l-fabp:Dbp-EGFP) larvae were fixed overnight with 4% paraformaldehyde in 1× PBS. Between seven and ten larvae per treatment were imaged as described above. Images were taken using a Nikon SMZ1500 stereomicroscope with a Nikon Digital Sight camera. Images were minimally processed using Adobe Photoshop CS4 and Adobe Illustrator CS4, were inverted using Adobe Photoshop CS4, and the fluorescence intensity was quantified using ImageJ.Tg(hand2:EGFP)pd24 larvae were treated and processed as described () by imaging on a Zeiss Pascal confocal microscope, and image processing and cell counting were conducted using Fiji (http://fiji.sc/Fiji). Prism 5.0c was used to plot all graphs and conduct statistical analyses, including Fisher’s exact test, one-way ANOVA or Student’s t-test as appropriate.Heatmaps of qPCR data were generated using GENE-E (Broad Institute, www.broadinstitute.org/cancer/software/GENE-E). Each square of the heatmap represents one data point for each gene and treatment. Colorization of the squares was determined via the median method in GENE-E. The median was calculated in the program for each row (gene) and subtracted from each data point. The resulting value was divided by the absolute deviation (AD) for the row. Data points were colored on a blue-white-red spectrum in which blue=3 ADs below the median or lower, white=median, and red=3 ADs above the median or higher. […]

Pipeline specifications

Software tools Adobe Illustrator, ImageJ, GENE-E
Applications Miscellaneous, Laser scanning microscopy, Microscopic phenotype analysis
Organisms Danio rerio, Caenorhabditis elegans, Drosophila melanogaster
Chemicals Ethanol, Hydrogen Peroxide, Oxygen