Fluorescein isothiocyanate fitc conjugated goat anti mouse igg

Unless otherwise noted, reagents were purchased from Sigma-Aldrich Chemie GMBH (Buchs, Switzerland). Antibodies (Abs) used for flow cytometry and/or immunofluorescence were anti- Vascular endothelial (VE)-Cadherin (BD Biosciences, Allschwil, Switzerland) and Alexafluor 488 conjugated anti- Platelet/endothelial cell adhesion molecule-1 (PECAM-1)/CD31 (Biolegend, USA), anti-JAM-B, anti-ICAM-1 (RND Systems, Abingdon, UK), anti-zonula occludens-1 (ZO-1), anti-Occludin-1 and anti-claudin-5 (Life Technologies Europe BV, Zug, Switzerland). Rabbit serum prior to immunization was used as a control (day 0) for labeling with polyclonal to human JAM-C (day 74) (Covalab, Lyon, France). Alexafluor 488 conjugated goat anti-rabbit IgG (Invitrogen) and Fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG (Jackson Immunoresearch, USA). FITC conjugated Anti-CD62L, and anti-CD14 (BD Biosciences). For histology and flow cytometry on Human umbilical vein endothelial cells (HUVECs), JAM-C studies were conducted using antibody 225.3, or a mouse anti-human IgG1isotype control (both produced in-house). For flow cytometry studies on HUVECs transfected with Small interfering RNA (siRNA), an anti-JAM-C antibody H33 conjugated to Alexa-488 was used. Labelling was done with a goat anti-human FITC or PE-conjugated secondary antibody (Jackson Immunoresearch, USA).

Cells were seeded on coverslips and when they reached 80% confluence, they were rinsed with PBS and fixed in 4% paraformaldehyde for 20 min, and then washed two times in PBS and treated in 0.3% TritonX-100 for 10 min for membrane permeabilization. Cells were then blocked with 4% BSA in PBST for 1 h at room temperature and incubated with the primary antibodies at 4 °C overnight, then washed five times in PBS and incubated with the secondary antibodies for 1 h at room temperature. Stained cells were washed five times in PBS and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Antibodies used were monoclonal mouse anti-Kaiso 6F/6F8-CHIP grade antibody (Abcam), polyclonal rabbit anti-Kaiso (H-154) antibody (Santa Cruz Biotechnology), monoclonal mouse anti-p120 Catenin antibody (BD Biosciences), phycoerythrin (PE)-conjugated goat anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). Microscopic pictures were captured under a Zeiss LSM-710 confocal microscope (Zeiss, Oberkochen, Germany).