Computational protocol: Lysine and Leucine Deficiencies Affect Myocytes Development and IGF Signaling in Gilthead Sea Bream (Sparus aurata)

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Protocol publication

[…] Cell proliferation was analyzed by immunostaining using a commercial PCNA staining kit (Cat. No. 93–1143, Life Technologies, Alcobendas, Spain) as related before []. Briefly, cells were washed and fixed at room temperature with 4% paraformaldehyde and postfixed with ethanol. Later, following the suggested manufacturer’s protocol, coverslips were blocked and incubated with anti-PCNA primary antibody and a biotinylated secondary antibody. Finally, cells were dehydrated in a graded alcohol series and mounted with histomount. The amount of PCNA-positive cells was calculated by dividing the PCNA-positive stained cells by the total number of nuclei in 14 images per coverslip containing a total of 400–1300 cells using the ImageJ software (National Institutes of Health, Bethesda, MD, USA). Digital images were acquired with a CC2 camera coupled to a microscope at 40X using analySIS (Soft Imaging System) software. All images were analyzed by the same researcher. [...] All statistical analyses were performed using the package IBM SPSS statistics v.20 (IBM, Chicago, IL, USA). Data was tested for normality using the Shapiro-Wilk test and for homogeneity of variances by Levene’s test. Differences through time within treatments were analyzed by a One-way ANOVA. Differences between experimental treatments at each time respect to the control condition were assessed by a Student’s t-test. When normality was not observed the non-parametric tests Kruskal-Wallis followed by Mann-Whitney U test were applied. In all cases results were considered statistically significant at p<0.05. […]

Pipeline specifications

Software tools ImageJ, SPSS
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Abramis brama, Sparus aurata, Danio rerio, Homo sapiens
Diseases Amino Acid Metabolism, Inborn Errors, Hyperlysinemias