Comprehensive Immunostaining for Immune Cell Characterization

Immunostaining was performed using the following antibodies: anti‐MPO for neutrophils (myeloperoxidase, A0398; Dako, Glostrup, Denmark; dilution 1:5000), CD68 for macrophages (clone PG‐M1, M0876; Dako; dilution 1:200), tryptase for mast cells (clone AA1, M7052; Dako; dilution 1:5000), EMBP (eosinophil major basic protein) for eosinophils (clone BMK‐13, MON6008‐1; MonoSan, Funakoshi, Tokyo, Japan; dilution 1:50); and CitH3 (citrullinated histone‐3) for ETs (ab5103; Abcam, Cambridge, UK; dilution 1:4000). NETs and METs were identified using sequential triple staining of anti‐CD68, MPO, and CitH3 (see supplementary material, Figure S1), whereas EETs and MCETs were visualised using sequential double staining of anti‐EMBP or tryptase with anti‐CitH3, respectively. Antigen retrieval was performed with heat‐induced antigen retrieval (Lab Vision™ PT Module; Thermo Fisher Scientific, Fremont, CA, USA) using Tris‐EDTA buffer (Thermo Fisher Scientific). For the secondary antibody, polymer horseradish peroxidase (HRP) anti‐rabbit or anti‐mouse (ImmunoLogic, Duiven, The Netherlands) was used and the immune complexes were detected using Vector Nova Red (Vector Laboratories, Burlingame, CA, USA) as chromogen. After each staining round, our sequential immunostaining protocol required the stained sections to be digitised using a slide scanner (Philips IntelliSite UFS; Philips Digital Pathology Solutions, Best, The Netherlands), followed by an elution step to remove the dye and immune complexes using stripping buffer, as previously described 6. Positive and negative controls (omission of the primary antibody) were always included (see supplementary material, Figure S2). Furthermore, control experiments for the staining of ETs consisted of pretreating the sections with DNase I (Thermo Fisher Scientific) as previously described 31.