Chicken anti rabbit af594

HUVEC pools were purchased from Invitrogen (Breda, The Netherlands). EGM-2 medium and SingleQuots^™ for HUVEC cell culture were purchased from Lonza (Verviers, Belgium). PAI-1 inhibitors PAI-039 (tiplaxtinin) and TM5275 were purchased from Axon Medchem BV (Groningen, The Netherlands) and diluted in DMSO (referred to as solvent). PAI-1 rabbit polyclonal antibody was a kind gift from Dr. Sacha Zeerleder. VE-cadherin monoclonal mouse antibody, clone BV6, was purchased from Millipore (Amsterdam, The Netherlands) and actin [AC-40] monoclonal mouse antibody was purchased from Sigma (Zwijndrecht, The Netherlands). GM130 rabbit monoclonal antibody was from Cell Signaling (Leiden, The Netherlands). Goat polyclonal anti-VE-cadherin [C-19] and rabbit polyclonal anti-α-catenin were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Mouse monoclonal antibodies directed against mouse monoclonal PECAM-1 AF488 [WM59], VE-cadherin AF 647 [55-7H1], IgG1 AF674, β-catenin [14 (link)] and p120 catenin [98/pp120] were purchased from BD Biosciences (Amsterdam, The Netherlands). Texas Red-X Phalloidin, chicken anti-goat AF647, chicken anti-rabbit AF488, chicken anti-rabbit AF594, chicken anti-mouse AF488 and IgG1 AF488 were from Invitrogen (Breda, The Netherlands).

Popliteal lymph nodes were placed in OCT compound, frozen on dry ice, and stored in -20°C until sectioning. 8 μm thick sections were blocked for 1 hour in 2.5% normal goat serum (Jackson ImmunoResearch) followed by incubation with directly conjugated (rat anti-CD3 AF647 clone 17A2 and rat anti-B220 AF488 clone RA3-6B2; Biolegend) and unconjugated polyclonal rabbit anti-collagen IV (Abcam) antibodies in goat serum over night at 4°C. Sections were then incubated with secondary antibody (chicken anti-rabbit AF594; Invitrogen) for 1 hour in goat serum and mounted in mounting media with DAPI (Vectachield, Vector Laboratories). Images were acquired with a confocal microscope (Zeiss LMS 800) and analysed using Fiji (ImageJ) software.