This figure represents the results of one representative experiment on cells originating from one kidney

f gingival histology of 12- and 24 Piclidenoson manufacturer week-infected and sham-infected mice demonstrating minimal gingival inflammation. P<0.05, P<0.01, P<0.001. doi:10.1371/journal.pone.0129795.g001 Gingival Inflammation The mandibles and maxillae of three mice from F. nucleatum-infected and sham-infected mice were fixed in 10% neutral buffered formalin, decalcified with Immunocal for 7 days. The decalcified tissue blocks were embedded in paraffin and sections were prepared at 5 m followed by staining with hematoxylin 4 / 19 F. nucleatum Repression of Inflammation in ApoEnull Mice and eosin. The stained slides were scanned at 20x using an APerio Scan Scope, and histological measurements were taken using APerio ImageScope v11.0.2.725 software. Inflammation was quantified by measuring the distance of gingival apical epithelial migration and counting the number of infiltrating leukocytes per 50m2 area of gingival tissue, by a reviewer blinded to the groups. Serum Antibody Analysis F. nucleatum-specific serum IgG and IgM antibody titers were determined by ELISA, using a standard ELISA protocol as described previously. Mean antibody titer values of infected mice were divided by mean antibody titer values of sham-infected mice, and the quotient PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19706235 represents the mean fold change in F. nucleatum-specific antibody titer due to infection. Graphs show mean fold-change in F. nucleatum-specific antibody titer of infected mice. Statistically significant differences between mean F. nucleatum-specific antibody titers of infected and sham-infected mice were determined by two-tailed Student’s t test, and are indicated above fold-change values on PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19705642 the graph. In vivo Localization of F. nucleatum by Fluorescence in situ Hybridization FISH was used to detect metabolically active bacteria. The protocol was performed as previously described. Slides were viewed at 63X with a Leica DMIRB microscope equipped with a Photometrics cascade-cooled EMCCD camera, controlled by the open-source software package Manager. Images were processed using Image J. Atherosclerotic Plaque Analysis Aortic arch and thoracic aortas were fixed in 10% neutral buffered formalin and embedded in paraffin. Sectioning and hematoxylin and eosin staining were performed as previously described. Plaque area, intimal layer thickness, medial layer thicknesses were measured and intimal/medial layer thickness ratios were calculated using ImagePro software by a blinded reviewer. Intimal/medial thickness ratios are used to reduce variations produced by differing vessel sizes. Quantification of Aortic Inflammatory Cell Infiltration Sections measured for atherosclerotic plaque were subsequently selected and stained for analysis of inflammatory cell infiltration. Each aortic section examined for atherosclerotic plaque was again evaluated for presence of CD3+ T cells and F4/80+ macrophages by immunohistochemical staining, as described previously. Serum Lipid Profile and SAA Level Blood was collected at euthanasia, and sera were separated by centrifugation. Thirty microliters of 24-week-infected mice and sham-infected mouse sera were analyzed by gel filtration high performance liquid chromatography analysis for lipid profiles. Levels of serum amyloid A of 24-week-infected mice and shaminfected mice were measured using a prepared ELISA kit from Kamiya Biomedical . Serum Nitric Oxide Measurement Serum NO concentrations were measured using a nitric oxide fluorometric assay kit from 24-week-infected and sham-infected mice. RT2 Prof