Structured subcellular localization of ferritin in proximal tubule brush border cells. (A) Immunofluorescence of kidney cortex sections using antibodies against H ferritin (green) and villin (red). Nuclei were stained with DAPI (blue). H ferritin and villin showed colocalization near the brush border region of proximal convoluted tubule cells. Ferritin concentration decreased toward the basolateral membrane of the cell and distribution showed a punctate pattern. Vesicle-like structures are visible at the basolateral membrane (arrowheads). Villin is an actin-binding protein found exclusively in the brush border of renal and intestinal epithelial cells. Experiments with an L-subunit–specific antibody gave similar results. Image visualization was made on a Nikon Eclipse E600 using a 63× magnification. CY3 and FITC were used as fluorochromes. Images were captured using a Nikon Digital Camera DXM1200F with Nikon ACT-1 Version 2.62 software. Images were processed with Adobe Photoshop. (B) Kidney cortex EM: Electron-dense particles of 5 to 7 nm in diameter that have clear characteristics of ferritin–iron cores are abundant in the proximal tubule brush border. Kidney cortex specimens were fixed with 2.5% glutaraldehyde and reduced osmium, followed by dehydration and embedding in Epon resin. Finally, microtome sections were mounted on copper grids and stained with bismuth subnitrate before visualization on a Technai T12 TEM at 120keV (FEI). Image acquisition was done using a 2k CCD ultrascan CCD camera (Gatan). Arrows denote electron dense ferritin particles.