Immunofluorescence Microscopy

HMVEC were grown to confluence on LabTek II chamber slides (Nunc, thermofischer scientific, Waltham, MA) that had been precoated with collagen and then treated for 6 hours with LPS in the presence and absence of catecholaminergic agents, and antagonists and agonists of α- and β-AR. Cells were then fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (PBS). After blocking with 1% bovine serum albumin in PBS, cells were incubated with either anti–zona occludens (ZO)-1-AlexaFluor594 (Invitrogen), F-actin (rhodamine phalloidin; Invitrogen), or anti–vascular endothelial (VE)-cadherin (Santa Cruz Biotechnologies) followed by AlexaFluor488-tagged secondary antibody. Nuclei were counterstained with 4′6-diamidino-2-phenylindole. Slides were visualized with a Cytation 5 Biotek (Fischer scientific, Waltham, MA) and recorded at 20X objective on Gen5 Image+ Software (Biotek,Winooski, VT).