2.10. Scanning electron microscopy (SEM) of fibrin clots

Fibrin clots were induced to form directly in a microwell plate by addition of 1 μL human thrombin (3 NIH U/mL final concentration) to 33 μL plasma from either the patient or a healthy donor, and the clotting mixture was incubated at room temperature for 120 minutes. The clots were washed 3 times with 0.1 mol/L PBS (pH 7.4) and fixed with 3% glutaraldehyde in the same buffer for 4°C overnight, then dehydrated in a graded series of ethanol solutions (50%, 70%, 80%, 90%, and 100% concentrations) twice. After the 100% ethanol step, samples were immersed in hexamethyldisilazane 3 times, followed by air-drying overnight in a fume hood. The clots were mounted on aluminum stubs and sputter-coated with 10 nm gold. Fibrin clots were examined on field emission scanning electron microscope (Tescan, Brno, Czech Republic), as described previously.^[5]