Rabbit anti mpo

5×10⁴ isolated PMNs seeded on poly-L-lysine-coated glass coverslips (BD Biosciences) in tissue-culture wells and allowed to settle prior to stimulation as described above. Coverslips were rinsed with ice-cold HBSS and the cells fixed with 4% paraformaldehyde and blocked overnight (HBSS with 10% goat serum, 1% BSA, 0.1% Tween20, and 2 mM EDTA) at 4°C. NETs were detected with rabbit anti-MPO (Dako) and rabbit anti-citrullinated histone H3 (citH3, Abcam). Secondary antibodies were goat anti-rabbit IgG AF555 and goat anti-rabbit IgG AF488 (Invitrogen). DNA was stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma) and NETs were visualized using a Zeiss Axioplan 2 Imaging fluorescence microscope in conjunction with a Zeiss AxioCam MRm monochromatic CCD camera and analyzed with Axiovision 4.8.2 software (Carl Zeiss).

For immunofluorescent double staining of MPO/properdin and C3c/properdin, 4µm thick liver sections were rehydrated and antigen binding sites were retrieved using preheated (95°C) Antigen Retrieval Buffer (Dako) for 30′, followed by a cooling down period of 20′. Non-specific binding sites were blocked with 10% goat serum in PBS. Sections were incubated overnight with rabbit-anti-MPO (Dako; 1∶1000) and monoclonal anti-C3c (1∶200) or goat-anti-properdin (Nordic Immunology, 1∶250) and monoclonal anti-C3c (1∶200) at 4°C, rinsed with PBS, and subsequently incubated with Alexa Fluor 488-conjugated donkey anti-goat (Invitrogen Molecular Probes, Eugene, OR, 4µg/ml) for 1,5 h at room temperature, followed by an appropriate Cy3-conjugated IgG antibody (Invitrogen, 1∶500) for 1,5 h at room temperature. Nuclei were stained with 4′,6-diamino-2-phenyl-indol (DAPI), and sections were mounted with Fluorescent Mounting Medium (Dako) and observed with a Leica immunofluorescence microscope.

Neutrophils obtained from the peripheral blood of healthy volunteers, from SFs of JIA patients, or from mice were placed on coverslips as described above. Cells were stained with rabbit anti-DEK antibody (1:100) or mouse anti-DEK antibody (1:50 or 1:500, BD Bioscience, 610948), mouse monoclonal anti-elastase (1:500, Abcam, Cambridge, MA, USA ab78187), rabbit anti-elastase (1:1,000, Abcam, ab 21595), mouse anti-LL-37 (1:100, Abcam, ab64892), or rabbit anti-MPO (1:500, Dako A0398) at room temperature for 1 h followed by incubation with AlexaFluor 488 goat anti-rabbit or AlexaFluor 594 goat anti-mouse antibody (Invitrogen). Nuclei and NETs were visualized by DAPI—Prolong gold antifade (Invitrogen P-36931) or stained with Hoechst. Slides were analysed using a fluorescence microscope (BX; Olympus) or confocal microscope (Nikon) including Z-stacks of 80 0.3 micron optical sections (× 60). Ten high-power (× 40) images were captured. Images were loaded onto Adobe Photoshop (Adobe System), and NETs were counted manually and shown as a percentage of total neutrophils per field. NETs were counted by at least three independent observers in a blinded fashion and identified based on overlap of DAPI staining with the NET markers elastase and MPO.