Ab5694

Tumor and renal fixed tissue sections (5 μ) were obtained to analyze tissue structure and fibrosis using classical hematoxylin-eosin and Masson staining, as well as to analyze the degree of angiogenesis, fibrogenesis and cell proliferation using the expression of CD31, α-smooth muscle actin (alpha-sma) and Ki67 by immunohistochemistry, respectively. Sections were deparaffinised, hydrated through graded ethanol steps, briefly rinsed in water and blocked at room temperature using TBSA-BSAT (10 mM Tris, 0.9% NaCl, 0.02% sodium azide, 2% bovine serum albumin and 0.1% Triton-x100 detergent). Slices were incubated overnight at room temperature with primary antibodies against 1:50 CD31 (Abcam, ab28364), 1:250 alpha-sma (Abcam, ab5694) and 1:500 Ki67 (DAKO, IR626) followed by incubation with the corresponding secondary antibodies either Alexa 488 Anti-rabbitt IgG (Invitrogen, A11008) or Alexa 488 Anti-mouse IgG (Invitrogen, A11001) for 5 hours diluted in TBSA-BSAT (1:500). Nuclear staining was performed using DRAQ-5^th (Red Fluorescen Cell-Permeable DNA probe, Biostatus Limited, United Kingdom). Immunofluorescence analysis was performed using Olympus BX61 microscope. Fluorescence quantification was performed using Leica Application Suite Advanced Fluorescence software and ImageJ software.

The human smooth muscle cells were prepared from explants culture, as previously described [18 (link)–20 (link)]. Briefly, primary smooth muscle cells were cultured from human saphenous veins from a similar cohort used for ex-vivo perfusion. Veins explants of 1–2 mm were plated, luminal side down, on the dry surface of a 24-well culture plate, previously coated with 1.5% Gelatin type B (Sigma-Aldrich). Explants were gently covered with one drop of RPMI, 10%FBS medium, and placed overnight in a 37°C, 5% CO2. The next day, culture medium was carefully added to the wells, taking care not to detach the explants. VSMC were identified by immunostaining using antibodies to smooth muscle actin (abcam, ab5694) and desmin (Dako, M 0760). Passages 1 to 4 were used for the experiments.