Immunofluorescent (IF) localization and quantitative analysis of heme-responsive gene 1 (HRG1/Slc48a1), a heme transporter, in the renal proximal tubules of mice during the neonatal period. (a) IF staining of HRG1/Slc48a1 (red channel) in the kidneys of mouse neonates, analyzed by confocal microscopy, reveals its apical and sub-apical localization in the epithelial cells of cortical renal tubules. Cell nuclei were counterstained with DAPI (blue). Scale bars correspond to 20 µm in the main images, and to 10 µm in the magnified insert. To confirm the specificity of HRG1/Slc48a1 detection, kidney sections were incubated only with the secondary antibody (negative control). (b) IF co-localization of HRG1/Slc48a1 (red channel) and aquaporin 1 (AQP1), a proximal tubule marker (green channel) in the kidneys of a 3-day old mouse. Cell nuclei were counterstained with DAPI (blue). Scale bars correspond to 20 µm. (c) Renal levels of HRG1/Slc48a1 assessed by Western blotting. The blot was reprobed with monoclonal anti-actin antibody as a loading control. (d) Immunolabeled HRG1/Slc48a1 and actin control bands were quantified using a Molecular Imager and the relative levels of HRG1/Slc48a1 (means ± S.D.) are plotted in arbitrary units (a.u). Results come from 2 separate blots and relative levels of proteins were standardized to a percentage with %S.D. in order to eliminate technical differences between blots. N values for each group are: 3dpp = 5, 5dpp = 5, 7dpp = 5, 9dpp = 5, 11dpp = 4. Data set for HRG1/Slc48a1 has normal distribution, therefore, one-way ANOVA was used (p = 0,0098, df = 4, F = 4,521). Tukey’s Multiple Comparison Test was used as post-hoc test. Small letters over the bars in the chart denote significant differences between age groups, with p < 0.05. dpp – days postpartum.