Zen 2.5 blue edition software

Image acquisition and analysis were performed at LiMiF https://limif.ulb.be/, the Light Microscopy Facility, Université Libre de Bruxelles, Faculty of Medicine, Campus Erasme, Brussels. IHC slides were observed on a AxioObserver Z1 inverted microscope (Zeiss, Jena, Germany), using a Plan Apochromat 20x/0.8 dry objective (Zeiss). Transmitted light illumination was provided by a HAL100 halogen lamp and condenser in “bright-field position”. Images (1920 by 1216 pixels, pixel size (x-y): 0.293 micron by 0.293 micron) were acquired with an Axiocam 702 monochrome camera (Zeiss) as proprietary *.czi files. Files were processed with Zen 2.5 (Blue Edition) software (Zeiss). Images were displayed in the linear mode with manual contrast adjustment and exported as 16 bits uncompressed TIF files.

Thrombus volume was measured via 3D reconstruction of the Z-stack image using Bitplane (IMARIS software version 9) as previously described [11 (link),12 (link)]. Briefly, a new surface was added to the Z-stack image via “add a surface” toolbar icon. The image contrast and brightness were adjusted, and the image was displayed in Blend mode. FITC was selected as the desired channel with absolute intensity set for thresholding. A threshold was manually adjusted to render all the fibrin structures. Finally, the volume of rendered thrombus was measured under the Statistics Tab. 2D images were analyzed with Zen 2.5 (Blue edition) software using identical settings between controls and irradiated cells (Carl Zeiss Microscopy, Germany). For analysis of AF-647 tagged antibody binding, the integrated density was determined per field of view using NIH Image J [33 (link)] after adjusting the threshold.