Mira 3 lmh

The fibrin network architecture was studied with scanning electron microscopy (Mira 3 LMH, Tescan Orsay Holding, a.s., Brno, Czech Republic). Fibrinogen from the patient and control samples was mixed with Ca²⁺ (8 mM, final concentration) and thrombin (final concentration 2 NIH U/mL) and incubated at room temperature for 3 h. The networks were then fixated with 4% formaldehyde overnight. The fixed samples were washed with PBS and water and subsequently dehydrated with a series of water–ethanol solutions with increasing ethanol concentration (30%, 50%, 70%, 80%, 90%, 95% and 2× 100%). Finally, the samples were dried using the CO₂ critical point method (Leica EM CPD300) and coated with 4 nm thick gold by sputtering (Leica EM ACE600). The fibrin networks were studied with an SEM, and the samples were methodically viewed to establish the consistency of the ultrastructure. Images were evaluated using ImageJ data analysis software (http://rsbweb.nih.gov/ij/; accessed on 13 May 2021). Fiber thickness and the average number of fibrin fibers per 1 micrometer square of fibrin clot were determined by analyzing ten different images captured from two independently prepared samples. Fiber thickness was determined from 100 values (10 measurements per image). The average number of fibers per 1 micrometer square was determined from 10 images per sample.

Fibrin net architecture was studied by scanning electron microscopy (Mira 3 LMH, Tescan Orsay Holding, a.s., Brno, Czech Republic). Thrombin (final concentration 2 NIH U/ml) was added to both modified and control fibrinogen and incubated at room temperature for 3 h. The networks were fixated by 4% formaldehyde and were washed with PBS and water and subsequently dehydrated with a series of water–ethanol solutions with increasing ethanol concentration (0%, 25%, 50%, 75%, and 100%). Eventually, the samples were dried using the CO₂ critical point method (Balzers CPD 010) and coated with 4 nm thick platinum by sputtering (Balzers SCD 050). Images were evaluated by ImageJ data analysis software (http://rsbweb.nih.gov/ij/) [63 (link)]. Fiber thickness and average number of fibrin fibers per 1 micrometer square of fibrin clot were determined by analyzing five different images captured from two independently prepared samples of both, modified and control fibrinogen. Fiber thickness was determined as mean ± SD from 200 values (20 measurements per image). Average number of fibers per 1 micrometer square was determined as mean ± SD from 10 images. The significance of differences was evaluated using student’s t-test. P values less than 0.05 (two-sided) were considered as statistically significant.