Mab clone 9 mouse anti human vimentin

ECs were plated on 8-chamber culture slides (BD Biosciences Discovery Labware, Milan, Italy) coated with 2 µg/cm² fibronectin (Roche, Milan, Italy). When cells grew to confluence, they were fixed and permeabilized with FIX & PERM (Società Italiana Chimici, Rome, Italy). Then cells were incubated with primary monoclonal antibody (mAb) (clone 9) mouse anti-human vimentin (Sigma-Aldrich), (cloneF8/86) mouse anti-human vWF (Dako-Cytomation, Milan, Italy), or mouse anti-human VE-Cadherin (kindly provided by Prof. Dejana from Institute of Molecular Oncology, Milan, Italy) (5 µg/mL) for 1 h at room temperature (RT) followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG for 1 h at RT. Images were acquired with Leica DM3000 microscope (Leica, Wetzlar, Germany) and collated using a Leica DFC320 digital camera (Leica).

ECs were detached from culture flasks with 5 mM EDTA at 37°C and a total number of 5 × 10⁵ were fixed with FIX & PERM cell permeabilization kit (Società Italiana Chimici) and incubated in permeabilization solution in ice for 30′ with mAb (clone 9) mouse anti-human vimentin (Sigma-Aldrich), mAb (cloneF8/86) mouse anti-human vWF, or mAb (cloneV9) mouse anti-human CK8/18. The binding of primary antibodies was detected by incubation with FITC-conjugated goat anti-mouse IgG. The membrane antigens were detected on unfixed cells, using monoclonal anti-human CD31, CD45, CD34, and CD105 directly FITC-conjugated, all purchased from Immunotools (Germany). The cells were fixed with 1% paraformaldehyde (Sigma-Aldrich) and analyzed for fluorescence with a FACScalibur instrument (BD Falcon, Milan, Italy) using CellQuest software.

EM cells were plated on 8-chamber culture slides (BD Biosciences Discovery Labware, Milan, Italy). Cells, when grown to confluence, were fixed and permeabilized with FIX & PERM (Società Italiana Chimici, Rome, Italy). Next, cells were incubated with primary monoclonal antibody (mAb) (clone 9) mouse anti-human vimentin (Sigma-Aldrich), (cloneF8/86) mouse anti-human vWF (Dako-Cytomation, Milan, Italy), or mouse anti-human CK 8/18 (Abcam, Italia, Milan, Italy) for 1 h at room temperature (RT), followed by fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG for 1 h at RT. Images were acquired using Leica DM3000 microscope (Leica, Wetzlar, Germany) and collated using a Leica DFC320 digital camera (Leica).