Metamorph offline software

Plasmodium falciparum strain 3D7 was cultured as previously described [11 (link)]. Blood from a healthy donor was collected in BD Microtainer Tubes containing K2E (K₂EDTA) (BD Biosciences, Franklin Lakes, NJ, USA). Giemsa microscopy was also performed as described earlier [11 (link)]. For automated counting of infected parasites, the parasite-infected RBCs were stained with SYTO21 (Thermo Fisher Scientific, Waltham, MA, USA) at a final concentration of 5 µM for 10 min. Bright field and fluorescence images of parasite-infected RBCs stained with SYTO21 were acquired using an inverted fluorescence microscope (DM1L, Leica Microsystems, Wetzlar, Germany) with a digital camera (MC120 HD, Leica Microsystems) and analysed using MetaMorph Offline software (ver. 7.8, Molecular Devices, Sunnyvale, CA, USA).

With the help of Stereo Investigator’s virtual tissue module, multiple images were acquired and stitched together in order to create one single image of the RECA-1 vessel stain in the whole cerebellum using a Leica light microscope at 10x magnification. All vessel morphology analyses where performed using the integrated morphometry analysis module in the Metamorph Offline software (Metamorph^®, Molecular Devices, Inc.). Each image was manually inspected, and a threshold was set to define the positive RECA-1 staining against the background. All analyses were measured in pixels and later recalculated into μm (1 pixel = 0.732 μm or 0.536 μm²). In this study, the following parameters were analyzed: ① total vessel surface area; ② total number of vessels; ③ vessel width; ④ vessel height; ⑤ vessel breadth; ⑥ vessel length; ⑦ mean radius; ⑧ vessel perimeter; and ⑨ shape factor: a value between 0 and 1 describing how closely the vessel approximates a circle, where a value near 0 indicated a flattened object and a value of 1 indicated a perfect circle. The shape factor was calculated using the following equation: 4πA/P², where P = perimeter and A = area.

Venous thrombosis in zebrafish larvae was assessed with two assays. The time-to-occlusion (TTO) was measured in seconds after laser injury laser of the posterior cardinal vein (PCV) in 3-day postfertilization (3 dpf) larvae [48 (link)]. In the second test, older, 5 dpf Tg(itga2b:EGFP) larvae with circulating fluorescent thrombocytes were subjected to a similar PCV injury and the thrombocyte fluorescence accumulation around the injury site recorded and measured. For both assays, larvae were anesthetized in Tricaine (170 μg/mL), placed on 0.22% low gelling point agarose on glass microscope slides and visualized with a Leica LMD microscope (Leica Microsystems, Wetzlar, Germany). The microscope used an HCX PL FLUOTAR L 20x/0.40 corr objective and a cryslas laser (max. pulse energy: 50 µJ, pulse frequency: 80 Hz, wavelength: 355 nm). Bright field images were acquired with a Leica LMD CC7000 camera and fluorescence with a Leica DFC 360 FX, using LMD and LAS-AF software, at room temperature. Thrombocyte activity image series were analyzed with MetaMorph Offline software, v.7.10.1.161 (Molecular Devices, LLC. San Jose, CA, USA). Thrombocyte numbers in 5 dpf larvae with the fga^+/+, fga^+/Δ19–56, fga^{Δ19–56/Δ19–56} genotypes were compared by counting itga2b:EGFP-positive cells flowing through the PCV over a 30 s period.