Two days post-confluence, endothelial monolayers on PA gels were treated with 0 and 1 μM simvastatin for 24 hours. Cells were fixed and permeabilized with 3.7% formaldehyde (VWR) and 1% Triton (VWR), respectively. VE-cadherin was visualized using a goat polyclonal VE-cadherin primary antibody (1:100) (Santa Cruz, No. sc-6458) and Alexa Fluor 568 donkey anti-goat secondary antibody (1:200) (Invitrogen, No. A11057). Fluorescent images were acquired on a Zeiss Axio Observer.Z1m microscope equipped with a Hamamatsu ORCA-ER camera using a 20x objective.
VE-cadherin junction width was quantified using ImageJ and a custom-written Matlab algorithm described previously [3 (link)]. Briefly, a line was drawn perpendicular to the cell junction in ImageJ to obtain a pixel intensity profile across each junction. The intensity profile was fit with a two-Gaussian curve in MATLAB and junction widths were defined as the width of the curve 20% above the baseline pixel intensity.
VE-cadherin junction width was quantified using ImageJ and a custom-written Matlab algorithm described previously [3 (link)]. Briefly, a line was drawn perpendicular to the cell junction in ImageJ to obtain a pixel intensity profile across each junction. The intensity profile was fit with a two-Gaussian curve in MATLAB and junction widths were defined as the width of the curve 20% above the baseline pixel intensity.