αKlotho trafficking in kidney and lung. A. Kidney. Red TAMRA dye-labeled FLAG-tagged αKlotho was injected intravenously as a bolus and surface nephrons were monitored by dual laser intravital fluorescent microscopy. Nuclei (green) highlight the tubules. B. At 30 min, kidneys were fixed and stained with anti-FLAG (red, for αKlotho) and phalloidin (green, for actin). Anti-aquaporin-2 (AQP2, blue) identifies collecting ducts. αKlotho was present in proximal tubules (PT) and distal tubules (DT) and not glomeruli (G). C. Exogenous FLAG-αKlotho tracked with time in the plasma and urine after a bolus intravenous injection. D. FLAG-αKlotho was injected into normal mice and the lungs perfusion-fixed and stained for FLAG-αKlotho (red), endothelium (CD31, green) and nuclei (SYTO, blue). Differential interference contrast (Top) and fluorescent microscopy (bottom) of the same image are shown [55]. 1 – intravascular. 2 – extravascular. 3 – non-vascular cells including type 2 alveolar epithelial cells.