[PubMed] [Google Scholar] 33. expression of ECM components, each of which shows distinct temporal patterns during AVF maturation. Increased expression of regulatory proteins such as MMP and TIMP precedes increased expression of structural proteins such as collagen and elastin, potentially mediating a controlled pattern of ECM degradation and vessel remodeling without structural failure. after circulatory flushing with PBS followed by 10% formalin. The tissue block was then embedded in paraffin and cut in 5-m cross-sections. Hematoxylin & eosin (H&E), Massons Trichrome, and elastic van Gieson (EVG) staining were performed on samples from preoperative as well as day 1 through day 42 AVF. Antibodies A mouse monoclonal antibody directed against mouse MMP-2 (Clone: 6E3F8) (ab86607) and rabbit polyclonal antibodies directed against mouse MMP-9 (ab38898), TIMP-1 (ab38978), and osteopontin (ab8448) were purchased from Abcam (Cambridge, MA). Antibodies against mouse collagen type I, III and fibronectin were raised in rabbits and purified as described elsewhere.(8,9) Immunohistochemistry The venous limb of the AVF was analyzed approximately 100 microns cranial to the fistula, i.e. between the fistula and renal vessels. Immunohistochemistry was performed using the Dako EnVision? + Dual Link System-HRP (Dako; Carpinteria, CA). For collagen type I, III, and fibronectin, sections were pre-treated with 3.0% hyaluronidase (bovine testicular origin, type I-S; Sigma-Aldrich Co, St. Louis, MO) in PBS (pH 7.4) for 30 min at 37C. For MMP-2, MMP-9, and TIMP-1, sections were heated in citric acid buffer (pH 6.0) at 100C for 10 min using Lab Vision PT Module (Thermo Scientific; Kalamazoo, MI). Imiquimod (Aldara) The sections were treated with 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activity and incubated with 5% normal goat serum in PBS (pH 7.4) containing 0.05% Imiquimod (Aldara) Triton X-100 (T-PBS) for 1 h at room temperature to block non-specific protein binding sites. Sections were then incubated at 4C with the primary antibodies diluted at 1:200 (anti-collagen type I, III, and fibronectin), 1:300 (anti-MMP-2), and 1:500 (anti-MMP-9, TIMP-1, and OPN) in T-PBS. After overnight incubation, the sections were incubated with EnVision reagents for 1 h at room temperature and treated with Dako Liquid DAB+ Substrate Chromogen System (Dako) to visualize the reaction products. Finally, the sections were counterstained with Dako Mayers Hematoxylin (Lillies Modification) Histological Staining Reagent (Dako). Relative quantification of histological and IHC staining was performed (MetaMorph, Molecular Devices, LLC, Sunnyvale, CA). Each venous limb of AVF sample was compared to respective sham vein sample of the same post-operative day where Nafarelin Acetate applicable and all samples were compared to controls stained simultaneously. Statistical Analysis All data was analyzed using Prism 6 software (GraphPad Software, Inc, La Jolla, CA). Comparison of AVF samples to paired sham samples as well as baseline pre-operative samples were made using paired t-test Imiquimod (Aldara) or one-way ANOVA with post-hoc analysis using Dunnetts multiple comparisons test where applicable. P values of .05 were considered significant. Microarray analysis was performed using hierarchical clustering analysis as well as principle component analysis and pathway enrichment analysis (MetaCore). Significant changes in gene expression of AVF compared to paired sham IVC were determined using a false discovery rate 0.05 and a fold change 2.0 or -2.0. Results Compared to preoperative veins, histological analysis of AVF revealed temporal changes in the vessel wall characteristics during the forty-two day study period (Physique 1ACC). The fistula thickened over.