Immunostaining indicates no change in ICAM-1 expression with substrate stiffness. ICAM-1 was measured as a function of HUVEC substrate stiffness using a fluorescently tagged antibody to ICAM-1 on nonpermeabilized TNF-α-activated HUVEC monolayers. Fluorescence images were taken over many locations on the nonpermeabilized HUVEC monolayer surface (A), and intensity (in arbitrary units, au) was quantified using ImageJ software (B). Scale bar in panel A is 20 μm and applies to all images. Substrate stiffness is indicated in the upper left corner of each image in panel A. Bars represent average of at least 20 images from each of 2 independent experiments. Error bars represent SE. ANOVA indicates that P > .05 among stiffnesses.