Mab3174

Immunohistochemical stainings with CD163 for macrophages (1:400 overnight 4° C, monoclonal rabbit anti‐CD163 [C‐terminal], Abcam, ab189915, EPR14643‐36, Cambridge, UK) and myeloperoxidase (MPO) for neutrophils (1:400 overnight 4° C, monoclonal mouse‐anti‐human‐myeloperoxidase antibody, R&D Systems, Inc., MAB3174, 392,105, Minneapolis, USA) as well as a routine toluidine‐blue‐staining for mast cell numbers (0.5% aqueous toluidine blue solution for 24 h) were performed from paraffin‐embedded lesional skin samples (MPO: UV = 36, CSU = 27; CD163: UV = 35, CSU = 27; Toluidine blue: UV = 20, CSU = 18), each section cut at 5 µm. An immunohistochemistry protocol with Polymer‐labelled secondary antibodies (anti‐mouse: EnVision+/HRP mouse, Dako, K400011‐2, Glostrup, Denkmark; anti‐rabbit: EnVision+/HRP rabbit, Dako, K4010, Glostrup, Denmark; both applied for 30 min at room temperature) and AEC + ‐substrate system (Dako, K346111‐2, Glostrup, Denmark, applied for 10 min at room temperature) was used for detecting the primary antibodies. For the immunohistological stainings, photos were taken by an Axioplan‐II‐microscope (Zeiss) at 200× magnification and % of positivity of stained area was assessed by Fiji software.

To stain for neutrophils and NETs, paraffin embedded sections of lungs were first de-paraffinized, and re-hydrated using standard protocols. Slides were boiled in Tris–EDTA buffer (pH = 9) for 8 min for antigen retrieval and not quenched for H202 afterward. Sections were then blocked with Fc Receptor blocker (#NB309, Innovex) for 30 min, and incubated with 1X donkey blocking buffer (5% donkey serum, 2.5% BSA, 0.1% Triton X-100 in PBS) for one hour. Mouse anti-human MPO (1:500, MAB3174; R&D Systems) and rabbit anti-human citrullinated histone H3 (1:250, ab5103; Abcam) antibodies were incubated together in 0.5X donkey blocking buffer at 4 C overnight. Then sections were incubated with Alexa Fluor 488 conjugated donkey anti-mouse (1:400, A21202, Thermo Fisher Scientific) and Alexa Fluor 568 conjugated donkey anti-rabbit (1:400, A10042, Thermo Fisher Scientific) secondary antibodies together in 0.5X donkey blocking buffer at room temperature for one hour. 4′,6-diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain (Thermo Fisher Scientific, D1306). Images were collected at 40 × magnification using a Leica TCS SP8 confocal microscope (40 × magnification) and were processed with Leica LAS X software.