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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\u201330\u2005min PFA fixation for the respective stainings. Our results provide a solid basis for future experiments to study neutrophil activation and NET-formation.<\/jats:p>","DOI":"10.1177\/17534259241307563","type":"journal-article","created":{"date-parts":[[2025,2,19]],"date-time":"2025-02-19T02:06:37Z","timestamp":1739930797000},"update-policy":"https:\/\/doi.org\/10.1177\/sage-journals-update-policy","source":"Crossref","is-referenced-by-count":6,"title":["The effect of chemical fixation with paraformaldehyde, glutardialdehyde  or methanol on immunofluorescence staining of neutrophils and neutrophil extracellular traps"],"prefix":"10.1177","volume":"31","author":[{"given":"Veronika","family":"Pilchov\u00e1","sequence":"first","affiliation":[{"name":"Institute of Biochemistry, University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, Germany"},{"name":"Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, 30559 Hannover, 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