Nb 200 579

For visualization of bacteria and NETs by immunofluorescence microscopy, 48 well plates and coverslips were prepared as described above in the NET induction assay. The samples were prepared with neutrophils, 10% autologous plasma and bacteria as described above in the neutrophil killing assay and co-incubated with methylprednisolone (625 µg/mL). Afterwards, centrifugation samples were incubated for 3 h (37 °C, 5% CO₂) and finally fixed with 4% paraformaldehyde. NETs were stained with a mouse monoclonal-antibody against DNA/histone 1 followed by an incubation with an ALEXA Flur488 Plus, as described above. Sc. canis was stained with a rabbit anti-Streptococcus suis antibody (self-made, 1:500 [109 (link),110 (link)]). St. pseudintermedius was stained with a rabbit anti-Staphylococcus aureus antibody (IgG: stock 4 mg/mL, Abcam ab20920, 1:100). E. coli was stained with a rabbit anti E. coli (IgG: stock 4.5 mg/mL, novus biologicals, NB 200-579; 1:100). As the secondary antibody, all bacteria were stained with a goat anti-rabbit Alexa 633-conjugated antibody (1:500; Thermo Fisher Scientific 2 mg, Waltham, MA, USA) for 1 h. The staining was finally conducted as described above. Respective isotype controls were used as described above.

Tissues were embedded in O.C.T. compound (VWR) and 5-μm-thick fresh cryosections on positively charged microscope slides (Superfrost/Plus; Thermo Scientific) were fixed with 4% paraformaldehyde or acetone-methanol (1:1 v/v). For H&E or immunohistochemistry, sections were blocked and permeabilized (0.2% Triton X-100, 5% goat normal serum (DAKO) or 1% BSA (Sigma), stained (anti-neutrophil antibody [NIMP-R14] (ab2557, Abcam), polyclonal E. coli antibody (1:100, NB200-579, Novus Biologicals), anti-IL-1 beta (1:50, ab9722, Abcam) or anti-MMP-7 (1:100, ab4044, Abcam), all rabbit antibodies). Alexa 488 anti-rat IgG or anti-rabbit IgG and Alexa 568 anti-rabbit IgG (A-21210, A-11001 and A-11011, Life Technologies) were secondary antibodies and nuclei were counterstained with DAPI (0.05 mM, Sigma-Aldrich). Imaging was by fluorescence microscopy (AX60, Olympus Optical). Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Scientific) were used to counterstain the tissue sections.
Histology was scored using H&E stained bladder sections. The score was based on neutrophil infiltration, tissue architecture and epithelial thickness on a scale of 0–10, where 0 is unchanged compared to uninfected controls and 10 the highest neutrophil infiltration, most destroyed tissue architecture and maximum epithelial thickness.