Mouse anti vimentin cy3

Antigen retrieval was carried out by heating the slides to 1258C in 90% glycerol (molecular grade) and 10% 0.01M citrate buffer pH 6.0 for 20 minutes in a pressure cooker. Sections were then briefly washed in water, incubated for 1 hour at room temperature in blocking buffer (1% BSA, 0.5% triton X-100 in PBS) and then in primary antibody (diluted 1:200 in blocking buffer) either at room temperature for 1 hour or overnight at 48C. Primary antibodies used were: rabbit anti-collagen IV (2150-0140; AbD Serotec, Kidlington, UK), goat anti-endoglin (AF1097; R&D systems, Minneapolis, MN, USA), mouse anti vimentin-Cy3 (C9080; Sigma-Aldrich, St. Louis, MO, USA), goat anti-GFAP (C9205; Sigma-Aldrich), mouse anti-alpha smooth muscle actin-Cy3 (C6198; Sigma-Aldrich). Sections were washed in washing buffer (0.1% tween20 in PBS) and incubated for 1 hour at room temperature in secondary antibodies (diluted 1:200 in blocking buffer; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). Subsequently sections were washed in washing buffer, treated with Hoechst (10 lg/ml in washing buffer) for 30 seconds, washed again and mounted in Moviol mounting medium (Sigma-Aldrich). Images were taken using a Leica DM IRB fluorescent microscope (Leica, Hicksville, NY, USA) and/or a Zeiss LSM700 confocal microscope (Zeiss, Jena, Germany).