Image j program

Measurements of VWF immunofluorescence were done using a × 40 Planapo objective (Leica) and a Leica (Leitz DMRB) fluorescent microscope equipped with a Leica DC380 digital camera. Cryosections from at least two different tissue blocks in each case were used. For quantitative analysis all sections were immunolabeled simultaneously using identical dilutions of primary and secondary antibodies and other reagents. Immunofluorescent images were obtained under identical parameters of imaging, zoom, pinholes, objectives, and fluorescence power. Sections exposed to PBS instead of primary antibodies served as negative controls. The image acquisition settings were standardized for all groups to ensure that the image collected demonstrated a full range of fluorescence intensity from 0 to 255 pixel intensity level and were kept constant during all measurements. Quantification of VWF was performed blindly, having on the screen only one channel showing F-actin labeling. For each patient at least 10 random fields of vision were analyzed using image analysis software (Leica) and Image J program as described [35] . Arbitrary units were calculated per unit surface area (AU/mm 2 ).