Axio observer z1m microscope

Two days post-confluence, endothelial cell monolayers on 10 kPa PA gels were treated with 0, 1, and 10 μM simvastatin for 24 hours. Cells were fixed and permeabilized with 3.7% formaldehyde (VWR) and 1% Triton (VWR), respectively. Immunostaining was done with a rabbit polyclonal cortactin primary antibody (1:100) (Santa Cruz, No. sc-11408) and Alexa Fluor 488 donkey anti-rabbit secondary (1:200) (Invitrogen, No. A21206). Actin was visualized with 594 FITC-conjugated phalloidin (1:100) (Invitrogen, No. A12381). Fluorescent images were captured on a Zeiss Axio Observer.Z1m microscope equipped with a Hamamatsu ORCA-ER camera using a 20x objective. Cell perimeters were outlined and cell circularity was calculated in ImageJ software where $circularity=\frac{4\pi \left(Area\right)}{{\left(Perimeter\right)}^2}$ , and a perfect circle has a value of 1. Cortactin organization was quantified using MatLab code to quantify the number of linear segments at cell-cell junctions.

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.

Endothelial cells were seeded on PA gels embedded with 0.5 μm diameter fluorescent beads (Invitrogen). Cells were allowed to adhere for 16 hours after which the media was removed and replaced with complete M199 containing 0 or 1 μM simvastatin. After 24 hours, phase contrast images of single cells were taken immediately followed by fluorescent images of the bead field beneath the cell. A second fluorescent image of the bead field in an unstressed state was taken after the cells were removed with trypsin/EDTA (Invitrogen). Images were acquired on a Zeiss Axio Observer.Z1m microscope equipped with a Hamamatsu ORCA-ER camera using a 20x objective. The magnitudes of traction force were calculated using the LIBTRC analysis library developed by M. Dembo (Dept. of Biomedical Engineering, Boston University) [38 (link)].