For the staining of ETs samples were permeabilized for 5 min (0.5% Triton X-100; Invitrogen) and blocked for 20 min (blocking buffer with 3% normal donkey serum, 3% cold water fish gelatin, 1% BSA, and 0.05% Tween20 in PBS). Then, the samples were incubated with a mouse monoclonal-antibody against DNA/histone 1 (4.4 μg/ml in blocking buffer, MAB3864; Millipore) for 1 h to visualize the ETs. The secondary staining was performed using a goat anti-mouse DyLight 488-conjugated antibody (1:500; Invitrogen). Staining with aqueous Hoechst 33342 (0.5 mg/ml) was performed for 10 min. After washing, all coverslips were embedded in ProLong® Gold antifade reagent (Invitrogen).
For the co-staining of ETs and neutrophil elastase within ETs, samples were prepared as described above. Then, the samples were incubated with a mouse monoclonal-antibody against DNA/histone 1 (4.4 μg/ml in blocking buffer, MAB3864; Millipore) for 1 h to visualize the ETs and a rabbit anti-neutrophil elastase antibody (1:25 in blocking buffer, Abcam #AB1876 [Cambridge, UK]) in blocking buffer. The secondary staining was performed using a goat anti-mouse DyLight 488-conjugated antibody (1: 500; Invitrogen) or a goat anti-rabbit Alexa 568-conjugated antibody (1: 500; Invitrogen). And stained aqueous Hoechst 33342 and embedded in ProLong® Gold antifade reagent as described above.
For the co-staining of ETs and neutrophil elastase within ETs, samples were prepared as described above. Then, the samples were incubated with a mouse monoclonal-antibody against DNA/histone 1 (4.4 μg/ml in blocking buffer, MAB3864; Millipore) for 1 h to visualize the ETs and a rabbit anti-neutrophil elastase antibody (1:25 in blocking buffer, Abcam #AB1876 [Cambridge, UK]) in blocking buffer. The secondary staining was performed using a goat anti-mouse DyLight 488-conjugated antibody (1: 500; Invitrogen) or a goat anti-rabbit Alexa 568-conjugated antibody (1: 500; Invitrogen). And stained aqueous Hoechst 33342 and embedded in ProLong® Gold antifade reagent as described above.