Digital images were captured using a Nikon DS 5M digital

camera and imported into Adobe Photoshop. When creating photographic plates for illustrations, brightness and contrast were adjusted for uniformity within a plate; no other alterations of images were done. Numbers of immunocytochemically identified cells were determined for neighboring pairs of 12 μm thick sections, one processed for F4/80 immunoreactivity and the other processed for albumin immunoreactivity. The sections were viewed with a 40× lens, in an area of 46,800 μm2 (260 μm × 180 μm), and photographed using fluorescein and ultraviolet filter sets. At least three different areas in each section were photographed and analyzed. In some cases, the two images for each set, taken with fluorescein and ultraviolet filter settings, were merged and counts were made find more of immunoreactive cells containing DAPI stained nuclei. In other cases, the nuclei could be identified as blank (dark) round or ovoid structures in the centers of the immunoreactive cells. Diameters of DAPI stained nuclei were measured using the Nikon DS-5M software for two point distances, or from Photoshop images, using a reticule. The average number of positive cells and standard deviation for each animal was calculated, and the overall mean number of cells with standard errors was calculated Pirfenidone for each cell type and age. The numbers of labelled cells (defined as an identifiable

nucleus amid immunoreactivity) in each defined area (260 μm × 180 μm) was adjusted by the formula presented by Abercrombie [33]: in which P is the calculated new average number of nuclei per region, A is the crude count of number of nuclei of labeled cells per section, M is the tissue section thickness (12 μm), and L is the average diameter of nuclei. Counts of numbers of labeled

cells did not differ between material with DAPI stained nuclei and unstained nuclei, so the data were combined. Acknowledgements Supported by NIH grant EB-003075 to KJL and grants from the UC Irvine Undergraduate Research Opportunities Program to BGL and to MST. References 1. Wisse E: An ultrastructural characterization of the endothelial cell in the rat liver sinusoid under normal and various experimental conditions, as a contribution to the distinction between endothelial and Kupffer cells. J Ultrastruct Res 1972, 38:528–562.PubMedCrossRef 2. Widmann JJ, Cotran RS, Fahmi HD: Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity. J Cell Biol 1972, 52:159–170.PubMedCrossRef 3. Wisse E: Observations on the fine structure and peroxidase cytochemistry of normal rat liver Kupffer cells. J Ultrastruct Res 1974, 46:393–426.PubMedCrossRef 4. Blouin A, Bolender RP, Weibel ER: Distribution of organelles and membranes between hepatocytes and nonhepatocytes in the rat liver parenchyma. A stereological study.