Anti histone h3 citrulline

To stain the NETs, neutrophils were fixed and stained with DAPI (4',6‐diamidino‐2‐phenylindole), anti‐Histone H3 citrulline (R2 + R8 + R17, ab5103), and anti‐MPO (Proteintech) followed by a goat antirabbit secondary antibody conjugated to Alexa Fluor 594 (Proteintech) and a goat antimouse secondary antibody conjugated to Alexa Fluor 488 (Proteintech). Thereafter, treated NETs were stained with anti‐Tissue factor antibody (Bioledgend) and anti‐MPO (Proteintech) followed by a goat antirabbit secondary antibody conjugated to Alexa Fluor 594 (Proteintech) and a goat antimouse secondary antibody conjugated to Alexa Fluor 488 (Proteintech), to visualize the coagulation factors binding to the NETs. Additionally, ECs cultured on fibronectin‐coated slide flasks were stained with anti‐VE‐cadherin antibody (ab33168) and DAPI or anti‐ZO‐1 antibody (Proteintech) and PI (propidium iodide). All the immunofluorescence images were analyzed through confocal microscopy.

To detect the phosphatidylserine (PS) exposure and CD62P(P‐selectin) of platelets, platelets isolated from healthy subjects and patients²⁶ were stained with fluorescein isothiocyanate (FITC)‐conjugated bovine lactadherin (Haematologic Technologies, Essex Junction, VT) and anti‐CD62P antibody (Proteintech). Thereafter, isolated neutrophils from patients and healthy donors were incubated with FITC‐conjugated‐CD41 (Bioledgend) and the PE‐conjugated‐CD66b (Bioledgend) antibody before analysis using a flow cytometer, to investigate the presence of neutrophil‐PLTs aggregates. Moreover, isolated neutrophils were fixed and stained with anti‐Histone H3 citrulline (R2 + R8 + R17, ab5103) and anti‐MPO (Proteintech), followed by a goat antirabbit secondary antibody conjugated to Alexa Fluor 488 (Proteintech) and a goat antimouse secondary antibody conjugated to Alexa Fluor 647 (ab150115), before analysis using a flow cytometer, to detect the NETting neutrophils.³⁶