Sp5 x inverted confocal microscope

Collagen
sheets were sterilized and human aortic endothelial cells (HAECs)
were trypsinized and seeded at density of 100,000 cells/cm² in fully supplemented EGM-2 onto collagen sheets or into individual
wells of 6-well plates and allowed to adhere for 48 h. Medium was
then replaced with fully supplemented EGM-2 without serum for 24 h
to achieve a quiescent phenotype. Positive control samples were treated
with TNF-α (100 ng/mL in EGM-2) for 4 h prior to fixation and
staining. Samples were fixed in buffered formalin (10%) for 20 min
at 4 °C, then washed three times with PBS pH 7.4 for 5 min each.
Permeabilization was completed with a 5 min incubation in Triton X-100
(0.3%) in PBS and samples were then washed three times with Triton
X-100 (0.1%) in PBS (PBS-T) for 5 min each. Non-specific binding was
blocked for 30 min at room temperature with a solution of Triton X-100
(0.1%) in PBS containing BSA (2%, Abdil) and then washed three times
with PBS-T for 5 min each. Primary antibodies were utilized at 1:50
dilutions (ICAM-1, VCAM-1 (Abcam)). Samples were also stained for
F-actin (1:40 from a 6.6 μM stock solution, Life Technologies)
for 1 h following standard protocol. Samples were mounted with Prolong
Antifade containing DAPI (Life Technologies) and stored at 4 °C
until imaging on a Leica SP5 X inverted confocal microscope (Wetzlar,
Germany).

Collagen
sheets were sterilized in ethanol solution (70%) containing an antibiotic/antimycotic
solution (1%) for 30 min and then rinsed with three washes of PBS
pH 7.4. vSMCs were trypsinized and seeded at a concentration of 40,000
cells/cm². Cells were allowed to adhere for 4 h and additional
media was then added to the tissue culture well. After appropriate
culture times, samples were stained with calcein AM (2 μM) and
ethidium homodimer (4 μM) and imaged with a Leica SP5 X inverted
confocal microscope (Wetzlar, Germany). Alignment was quantified using
a fast Fourier transform function in ImageJ on an intensity threshold
based binarized image, utilizing the radial summing profile in 5°
increments. These data were then plotted and FWHM calculated. Cell
shape index (CSI), a dimensionless measurement of cell morphology was quantified using the open source CellProfiler
image analysis software (https://cellprofiler.org) to determine the area (A) and the perimeter (P) of each cell, CSI was calculated as previously described.^{36 (link)}