Col 94

The following antibodies were used: anti-mouse β-catenin (mouse clone 14/beta-catenin, 1:100, BD Biosciences); anti-human β-catenin (rabbit polyclonal, 1:1000, Abcam); anti-mouse N-cadherin (mouse, clone GC-4, 1:200, Sigma); anti-mouse laminin (rabbit polyclonal, 1:100, Sigma); anti-human collagen-IV (mouse, clone Col-94, 1:300, Sigma); anti-human vimentin (rabbit, SP20 clone, human specific, 1:300, Thermo Scientific), anti-bovine glial fibrillary acidic protein (GFAP) (chicken polyclonal, 1:1000, Abcam); anti-human nestin (rabbit polyclonal, human specific, 1:100, Millipore). Primary antibodies were visualized with secondary AlexaFluor-conjugated goat-anti-mouse, goat-anti-rabbit or goat-anti-chicken polyclonal antibodies (Invitrogen; 5 µg/ml). For background controls, isotype-matched unspecific mouse IgG (BD Biosciences) was used for monoclonal antibody stainings, and for polyclonal antibody stainings samples were incubated only with secondary AlexaFluor-conjugated antibodies. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 2.5 µg/ml). F-actin was labelled with AlexaFluor-conjugated phalloidin (Invitrogen). Growth factor-reduced reconstituted basement membrane (rBM) (Matrigel, BD Biosciences; 9.8 mg/ml) was used for rBM interface culture.

Endothelial cell surface, basement membrane, and nuclei stained with FITC-labeled Ulex europaeus lectin (Vector labs), mouse anti-collagen IV [COL-94, (Sigma)]/anti-mouse Alexa 594 antibody, and DAPI respectively. Pictures were obtained on a Nikon TE200 fluorescence microscope using the Metamorph software package (Universal Imaging) and were process in ImageJ software.