Briefly, plasma was supplemented with 2 % (w/w) of human fibrinogen conjugated to Alexa Fluor 488 (Thermo Scientific). Clot formation was initiated with the addition of 1 U thrombin (EMD Millipore) in the presence of 2.4 mM calcium as described [31] (link).
For fibrin-vimentin interaction, plasma was supplemented with 2 % (w/w) of human fibrinogen conjugated to Alexa Fluor 647 (Thermo Scientific) and with mouse anti-human Vimentin (V9, Sigma-Aldrich) or mouse IgG (isotype control) prior to initiating clot formation with the addition of 1 U thrombin (EMD Millipore) in the presence of 2.4 mM calcium as described [31] (link). Fibrin clots were washed three times with phosphate buffered saline (PBS) and incubated immediately with a secondary anti-mouse IgG-CF488A (1:1000, Sigma) conjugate for 1 h. Then, fibrin-clot were washed three times with PBS and fixed with 4 % paraformaldehyde for 10 min and washed three times for visualization by confocal microscopy.
For fibrin-vimentin interaction, plasma was supplemented with 2 % (w/w) of human fibrinogen conjugated to Alexa Fluor 647 (Thermo Scientific) and with mouse anti-human Vimentin (V9, Sigma-Aldrich) or mouse IgG (isotype control) prior to initiating clot formation with the addition of 1 U thrombin (EMD Millipore) in the presence of 2.4 mM calcium as described [31] (link). Fibrin clots were washed three times with phosphate buffered saline (PBS) and incubated immediately with a secondary anti-mouse IgG-CF488A (1:1000, Sigma) conjugate for 1 h. Then, fibrin-clot were washed three times with PBS and fixed with 4 % paraformaldehyde for 10 min and washed three times for visualization by confocal microscopy.