In liver sections from FVIII-KO mice staining of both rFVIII (A and C, green) and rFVIIIFc (B and D, green) is intensely localized to Kupffer cells. In addition, rFVIII, but not rFVIIIFc, shows a distinct punctate staining associated with hepatocytes. Staining for LSEC and endothelium (A’ and B’, CD31, red) reveals the central vein (CV) and the diffuse sinusoidal network and confirms that the punctate staining of rFVIII (A’, green) is not associated with LSEC, while some rFVIIIFc staining (B’, green) localizes with the liver sinusoid. Nuclei are stained blue with DAPI (A’ and B”). Additional stainings confirm co-localization of rFVIII (C’) and rFVIIIFc (D’) in Kupffer cells (CD68, red). VWF (C” and D”, magenta) is also associated with FVIII staining in Kupffer cells (arrows), however VWF in Weibel-Palade bodies in the endothelial lining of the central vein is not associated with FVIII. For orientation in liver lobules: CV, central vein; S, sinusoid; KC, Kupffer cell and HC, plate of hepatocytes (scale bar, 20 μm).