MCA thrombosis. (A) Representative images of an occluding MCA thrombus formed in vehicle-treated guinea pigs at immunofluorescence. Sections of frozen brains were cut and stained for a platelet marker (CD41, red) and fibrinogen/fibrin (green) and were counterstained for nuclei (blue): thrombus is mainly formed by platelets with only minor presence of fibrin (A, Magnification ×20; B, Magnification ×100). (B) Positive control for fibrin: a thrombus obtained in the stenosis-induced deep venous thrombosis model was longitudinally cut and stained for fibrinogen/fibrin (green) and counterstained for nuclei (blue). Magnification ×100. A Zeiss Axiovert 200 inverted fluorescence microscope (objectives: Zeiss Plan-Apochromate 10×/0.45 or 63×/1.4) connected to a monochrome camera (AxioCam MRm) was used for imaging. Colors for fluorescent channels were assigned by using Axiovision software (Axio Vs 40, Version 4.6.3.0). (C-L) Representative histologic micrographs of MCA thrombus of guinea pigs treated with vehicle and ALX-0081 at 5 mg/kg after photochemical-induced thrombosis. Slides were stained with hematoxylin and eosin for cellular components (top row), or phosphotungstic acid for fibrin (bottom row). (C,H) Vehicle-treated guinea pigs (brains collected immediately after the complete occlusion of MCA). (D,I) Vehicle-treated guinea pigs (brains collected 1 hour after the complete occlusion of MCA). (E,J) ALX-0081 administered at the moment of complete occlusion. (F,K) ALX-0081 administered 15 minutes after occlusion. (G,L) ALX-0081 administered 60 minutes after occlusion. Sections were analyzed by optical microscopy (Leica; Wetzler, Germany) using ×40 magnification.