Abstract
The formation of neutrophil extracellular traps (NETs) is known as an important part of the innate immune response. Still, some mechanisms regarding their formation and role during a disease are not completely understood yet. To visualize NETs by immunofluorescence microscopy, a chemical fixation is required. Therefore, this study focused on the effect of chemical fixatives on immunofluorescence staining of selected neutrophil and NET-markers, including myeloperoxidase (MPO), DNA/histone-1-complexes and citrullinated histone H3 (H3cit). Neutrophils isolated from fresh human blood were stimulated with phorbol-12-myristate 13-acetate (PMA) to induce NETs and fixed with paraformaldehyde (PFA, 4%), glutardialdehyde (GA, 5%) or methanol (MeOH, 100%) using different incubation times depending on the used fixative. We found that different fixation times with PFA had no effect on the staining intensity of MPO or DNA/histone-1-complex antibodies. For the staining of H3cit, fixation with PFA for 24 h decreased the signal intensity whereas 30 min fixation time had no effect. In contrast, glutardialdehyde induced a high amount of autofluorescence, and the fixation with 100% MeOH resulted in visible cellular damage. Therefore, we recommend 15-30 min PFA fixation for the respective stainings. Our results provide a solid basis for future experiments to study neutrophil activation and NET-formation.