4 6 diamidino 2 phenylindole dihydrochloride

Immediately after the T-TAS analysis, immunofluorescence analysis was performed to determine the composition of thrombi formed in the flow chamber [22 (link)]. Platelets in unfixed thrombi were labeled with FITC-conjugated mouse anti-human CD41 IgG (Beckman Coulter, Miami, FL, USA) for 15 min in the dark. After fixation with OptiLyse C (Beckman Coulter), fibrin (ogen) was detected by using rabbit anti-human fibrinogen IgG (Dako, Tokyo, Japan) labeled with Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA) for 30 min in the dark. The nuclei of leukocytes were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (Dojindo, Kumamoto, Japan). The entire image of thrombi formed in the flow chamber was analyzed with a BZ-X700 All-in-One Fluorescence Microscope (Keyence Corp., Osaka, Japan). Although fibrin generation could continue until fixation with OptiLyse C, this had little impact on the results.

The filter was fixed with OptiLyse C (Beckman Coulter, Marseille, France) for 15 minutes and then treated with phosphate-buffered saline (PBS) for 5 minutes. After three washes with PBS containing 0.1% triton (PBST), unspecific binding sites were blocked by incubation with PBST containing 1% bovine serum albumin (Sigma-Aldrich) for 60 minutes. The filter was then incubated with 60 µg/mL rabbit anti-fibrinogen antibody (Agilent Dako, Santa Clara, CA, USA) in PBST containing 1% bovine serum albumin for 60 minutes. After three washes with PBS, the filter was incubated with 20 µg/mL goat anti-rabbit antibody conjugated with Alexa Fluor 594 (Life Technologies, Eugene, OR, USA) for 60 minutes in the dark. After washing, the filter was incubated with 2 µM 4′,6-diamidino-2-phenylindole dihydrochloride (Dojindo, Kumamoto, Japan) for 5 minutes in the dark. Whole images of the filters were analyzed with the All-in-One Fluorescence microscope BZ-X700 (Keyence Corp., Osaka, Japan) and details were analyzed with the confocal microscope LSM 700 (Zeiss, Oberkochen, Germany).