Immunofluorescence Analysis of Neutrophil Extracellular Traps

The above treated neutrophils were fixed by 4% paraformaldehyde in PBS overnight at 4 °C, then permeabilized with 0.1% TritonX-100 in PBS for 20 min, and washed three times with PBS. Cells were blocked with 1% BSA, 0.1% Tween 20 in PBS for 2 h at room temperature, and then incubated with rabbit anti-MPO antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4 °C. Next, neutrophils were gently washed three times with PBS and incubated in the dark with goat anti-rabbit IgG antibody (Alex Flour 647 nm, 1:500 diluted in the PBS) for 2 h at room temperature. Before staining with 100 ng/ml DAPI, neutrophils were incubated with rabbit anti-CitH3 antibodies (Abcam, UK, 1:200 diluted in 0.1% Tween 20 in PBS) overnight at 4 °C, then washed three times and incubated with Alexa fluor 488 nm goat anti-rabbit IgG fluorescent secondary antibody, finally washed with PBS and ProLong Diamond Antifade Mountant (Thermo, USA) was added to each coverslip, the coverslips were placed on the slides, and then observed with a confocal fluoresce microscope (FV3000 Olympus, Japan). Images were analyzed with the ImageJ software from National Institutes of Health^{33 ,61 (link)}.