Figure 7. Immunohistochemical staining for PPARc and the cell proliferation marker Ki-67 in uninjured and injured carotid arteries. A, Paraffin-embedded, uninjured and injured aortae of the indicated genotypes were cut into 6 mm sections for immunohisto- chemistry, with PPARc positive media and neointimal cells staining dark brown (arrowheads) in methyl green counterstained sections. B, Percentage of arterial media and neointimal PPARc positive cells, determined as described in Methods. C, Immunohistochemical detec- tion of Ki-67 in representative WT and Vnn12/2 carotid sections. D, Percentage of arterial media and neointimal Ki-67 positive cells. Data are mean 6 SD of 3 independent experiments. *P,0.05 WT vs. Vnn12/2. doi:10.1371/journal.pone.0039106.g007 Vanin-1 in Vascular Remodeling Disease (Invitrogen, Carlsbad, CA). Peroxidase activity was demonstrated by exposing sections to the substrate, 3,39–diaminobenzidine tetrahydrochloride (DAB) and counterstaining with 1% methyl green. For negative control sections, PBS was substituted for the primary antibody. We calculated percentage of positive staining SMCs in media and neointima (relative to total cell number) in 5 different 20 X magnification fields in each section, studying 8 sections from each control and ligated sample from each mouse. SDS-PAGE/Western Blot Analysis SMCs were lysed in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 10 mg/ml leupeptin, 1 mg/ml aprotinin). Protein extracts (35 mg) were separated by 10% SDS-PAGE, and transferred to Immobilon-P membranes, and, after blocking, blots were incubated with primary antibodies for 18 h at 4uC. The membrane was washed, and then incubated withrespective secondary antibody conjugated with horseradish peroxidase. Enhanced chemiluminescence re- agent (Thermo Scientific, Rockford, IL,) served as substrate solution, used according to manufacturer instructions. Horizontal scanning densitometry of films was performed using universal hood densitometry (Bio-Rad, Hercules, CA). Assay of MMP Activity Gelatinase activity was determined by zymography as previ- ously described [42,43]. Equal amounts of conditioned media from identical numbers of cells, grown under serum free conditions for 48 h, were loaded onto 10% SDS-polyacrylamide gels containing 0.1% gelatin, and zymography was performed. After electrophoresis, gels were incubated for 45 min in renatur- ation buffer and in developing buffer (Invitrogen, Carlsbad, CA) for 24 h at 37uC. Gels were stained with Coomassie blue; clear bands indicated active enzymes (MMP-9 pro-form, 92 kDa; active form, 83 kDa). Enzymatic activity was estimated by densitometry of negative-image zymographic gels and measured in OD units. Cell Proliferation and Migration Studies We used the Invitrogen non-radioactive Cell Proliferation Assay kit, per manufacturer instructions, to determine cell proliferation. SMC migration was assayed using the Transwell system (Corning, NY), with a polycarbonate membrane in six-well plates. SMCs were plated at a concentration of 1.0x 106 cells/ml. Numbers of cells migrated per well, after 48 h treatment with PDGF or diamide, were determined by thiozolidine bluestaining. Images were analyzed using a microscope with digital camera (Advanced Microscopy Group, Bothell, WA). Statistical Analyses Unless otherwise indicated, data are presented as the mean 6 SD of determinations from 3 or more experiments. Results were compared by one-way ANOVA followed by Bonferroni multiple- comparison analysis. A value of p,0.05 was used to define statistical significance. Author Contributions Conceived and designed the experiments: KJD RT. Performed the experiments: KJD SS. Analyzed the data: KJD SS RT. Wrote the paper: KJD RT. PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e39106