Chicken anti rabbit af488

Primary antibodies included the following: EEA1 (Santa Cruz Biotechnology, sc-6415), Rab7 (Abcam, ab 137029), Lamp1 (BD Pharmingen, 1D4B), GFP (Life Technologies, A11122). Secondary antibodies included the following: donkey anti-goat–AF555 (Invitrogen, A21432), goat anti-rat–AF488 (Invitrogen A11006), chicken anti-rabbit–AF488 (Invitrogen, A21441).
Other reagents included the following: AF594/Fab (Jackson ImmunoResearch Laboratories Inc., 309-586-003), A488 phalloidin (Life Technologies, A12379), AF555 phalloidin (Life Technologies, A34055), DQ Green BSA (Life Technologies, D12050), Tf-AF647 (Life Technologies, T23366), dextran-AF488 (Life Technologies, ID7178), WGA-AF647 (Life Technologies, W32466).

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).

Membranes were stained as described above with additional AnnexinV-BV421 staining (Milteny Biotec). Subsequently, cells were fixed and permeabilized by a permeabilization buffer set (eBioscience) with 1% paraformaldehyde, 0.5% saponin and stained with either rabbit anti-Mx1 (ProteinTech, Chicago, USA), rabbit anti-MDA5 (Abcam, Cambridge, UK), rabbit anti-DDX58 (Abcam), rabbit anti-IFI16 (Abcam) and rabbit anti-ZBP1 (Thermofischer, Rockford, USA)) and incubated in the dark for 45 min on ice. As a secondary antibody, chicken anti-rabbit-AF488 (Invitrogen, Carlsbad, USA) was used. Unstained cells and isotype-matched controls (Becton Dickinson Biosciences) were used to assess antibody specificity. Analysis was performed as previously described [21 (link)].