Immunofluorescent (IF) localization and quantitative analysis of ferroportin (Fpn) in the kidneys of neonatal mice combined with quantitative expression analysis of Hamp gene in livers of experimental mice. (a) IF staining of Fpn (red channel) in the epithelial cells of cortical renal tubules of mouse neonates, analyzed by confocal microscopy. A positive signal is visible on the basolateral membrane of renal cortex epithelial cells in 3- (magnified insert), 5- and 7-day-old mice. Cell nuclei were counterstained with DAPI (blue). To confirm the specificity of Fpn detection, kidney sections were incubated only with the secondary antibody (negative control). Scale bars correspond to 20 µm in the main images, and to 10 µm in the magnified insert. (b) Quantitative analysis of the Fpn fluorescent signal in renal cortical tubules. The mean fluorescence signal associated with Fpn in each tubule section was measured by ImageJ analysis (3 individuals per age group, 10 measurements per individual, N = 30 per each age group) and quantified manually as a Mean Grey Value, the intensities (means ± S.D.) are plotted in arbitrary units (a.u). Data set for Fpn is not normally distributed, therefore, nonparametric, Kruskal-Wallis ANOVA was used (p < 0.0001, df = 4, H = 125,4). Dunn’s Multiple Comparison Test was used as post-hoc test. (c) Basolateral Fpn localization in the initial segment of the nephron (arrow) reveals that this protein is present in the proximal renal tubules. Cell nuclei were counterstained with DAPI (blue). The scale bar corresponds to 20 µm. (d) RT-qPCR analysis of Hamp transcript levels in the livers of mouse neonates from 3 to 11 days old. The histogram displays relative Hamp mRNA levels in arbitrary units (means ± S.D.). N values for each group are: 3dpp = 4, 5dpp = 5, 7dpp = 4, 9dpp = 4, 11dpp = 4. Data set for Hamp expression is not normally distributed, therefore, nonparametric, Kruskal-Wallis ANOVA was used (p = 0,0053, df = 4, H = 14,74). Dunn’s Multiple Comparison Test was used as post-hoc test. Capital and small letters over bars in the charts (b,d) denote significant differences between age groups, with p < 0.01 and p < 0.05, respectively. dpp – days postpartum.