Quantification of the Association of Cy3-Labeled VLP with Liver Cells

Based on 4-color immunofluorescence analysis of liver sections, we determined that Cy3-labeled VLP associated with LSEC and KC, but not hepatocytes or large vessel endothelium. The association of VLP with LSEC and KC was quantified with Image J software as described previously in our work (1). Briefly, quantification was determined in a three-step process: (1) image threshold was adjusted to account for background intensity not associated with Cy3 VLP, and the total Cy3 fluorescence intensity was recorded for the image; (2) each Kupffer cell (KC) within the image, identified by anti-F4/80 staining, was partitioned, and the fluorescence intensity of Cy3 associated with individual KC was recorded; the VLP intensity associated with individual KC was summed to total KC-associated VLP; and (3) the total KC-associated VLP intensity was subtracted from the total VLP intensity of the entire image to give the total LSEC-associated VLP intensity. KC and LSEC association was averaged for a total of 160 technical replicates of the images over the three mice. Within each mouse, the technical replicates were averaged producing three KC and three LSEC associations. The mean and SD of the three KC- and the three LSEC-association observations are presented and no statistical testing was done since these observations are not independent of each other.