Computational protocol: Immunofluorescence identifies distinct subsets of endothelial cells in the human liver

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[…] Sections were blocked with 0.25% casein for 10 minutes at room temperature, then incubated with the primary antibodies listed in for one hour at room temperature. The primary antibodies were detected with the corresponding isotype-specific goat anti-mouse or goat anti-rabbit secondary antibodies conjugated to a fluorochrome (488, 555 or 647; Invitrogen, CA, USA). DAPI was included at 0.0005% w/v with secondary antibody. Following staining, background autofluorescence was quenched by bathing slides in 0.1% Sudan Black B Solution for 15 minutes followed by two brief washes in TBS then three 1 hour washes in TBS. All data shown of liver tissue are representative of at least 3 or more biological replicates and were consistent findings among all livers assessed. The number of bioligical replicates in which each antigen was assessed in liver tissue is presented in . Data of positive control tissue presented in the was performed in one biological replicate.The slides were mounted using Prolong Gold (Invitrogen). Sections were visualized with a Nikon Ni-U fluorescent microscope equipped with the epi-fluorescent filters: UV, 450–490 nm, 530–560 nm and 590–650 nm. Images were acquired at room temperature using 4x/0.13 numerical aperture (NA), 10x/0.45 NA, 20x/0.75 NA and 40x/0.95 NA Nikon objectives, a SPOT Pursuit 1.4MP camera and Spot v.5.0 Spot software (Sterling Heights, USA). Images were generated using Cytosketch (CytoCode, Auckland, New Zealand) and figures were formated using Adobe Illustrator (Adobe Systems, Mountain View, CA). [...] Quantifying differences in cell number and marker expression is a highly complex process using immunofluorescence, however, in order to provide quantified evidence of observed differences in the distribution of fluorescence of LSEC markers (CD36, CD32, CD14 and LYVE-1) between Z1 and Z3, ImageJ (National Institutes of Health, Bethesda, Maryland, USA) was used to assess the mean intensity of fluorescence in a greyscale image in 5 randomly chosen Z1 areas and 5 randomly chosen Z3 areas of the same size in images from 3 different patients. For each liver, the mean intensity of fluorescence was assessed in different zones of the same image (to ensure the same conditions of immunostaining, visualization and image capture).To quantify intensity of expression for PT and CV makers ImageJ was again used to assess the the highest level (or “Max”) intensity of fluorescence in 5 different PTs and 5 different CVs within the same image(again to enure the same conditions of immunostaining, visualization and image capture). This was repeated for 3 livers.A linear mixed model (LMM), accounting for multiple measurements made on each liver, was fitted to the log-transformed intensity data for each marker (CD14, CD36, CD32, LYVE-1 in the case of LSEC markers and CD146, aSMA, and laminin in the case of PT and CV markers). The log-transformation was required to meet the underlying assumption of homogeneity of variance of the LMM. The results from the pairwise comparisons of means between zones (or PT and CV) were back-transformed to their original scales and are reported as ratios, this being the median intensity of Z3(or PT) relative to the median intensity of Z1(or CV). Also reported are 95% confidence intervals for these ratios and p-values resulting from the t-tests.The results of the quantative and statistical analysis are reported in the (see and ). […]

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