Eclipse ti u 100

Frozen lung tissue samples of rats were sectioned at 7 µm intervals and fixed with 4% paraformaldehyde. For the detection of MPO-positive cells (neutrophils, monocytes), sections were permeabilized with 0.2% Triton X-100 and incubated with 5% normal goat serum + 0.5% BSA + 0.2% TritonX-100 in PBS in order to block unspecific binding. Sections were further incubated with anti-MPO antibody (1:100, Abcam) for 1 h followed by additional incubation with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:1000, Cell Signaling Technology) overnight at 4 °C.
For the detection of leukocytes, a mouse monoclonal anti-rat CD45 antibody was used (clone OX-1, 1:20, BD Pharmingen). Then, slides were further incubated with Alexa Fluor 555-conjugated goat anti-mouse IgG (H + L) secondary antibody (1:1000, Thermo Fisher) for 1 h at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenyl indole (DAPI) and slides were mounted in fluorescence mounting medium (Dako). Tissue sections were examined using an inverted microscope (Eclipse Ti-U 100, Nikon). Signal intensity of CD45 immunolabeling in ten random fields was quantified as mean value and averaged using the Image J analysis software.

Freshly isolated human or murine bone marrow-derived neutrophils (1.5 × 10⁵) were seeded on polylysine-coated glass coverslips (Ø 18 mm), allowed to settle for 30 min, and then treated with 100 nM PMA. After 3.5 h of activation, cells were fixed with 4% paraformaldehyde (PFA), blocked with 5% normal goat serum (Cell Signaling Technology) and 1% bovine serum albumin (BSA) (Carl Roth) and incubated with polyclonal antibodies against myeloperoxidase (MPO) (Abcam) for 2 h at room temperature. Cells were further incubated with a secondary anti-rabbit Alexa Fluor 488-conjugated antibody (Cell Signaling Technology) for 1 h, counterstained with DAPI and mounted in Dako fluorescent mounting medium (Dako). Cells were examined with an inverted microscope (Eclipse Ti-U 100, Nikon).

To induce NETosis, freshly isolated bone marrow-derived neutrophils (1.5 × 10⁵) were seeded on poly-L-lysine coated coverslips. Cells were stimulated with 4 μM ionomycin for 3 h. After that, cells were fixed with 4% PFA, blocked and incubated with a polyclonal rabbit anti-myeloperoxidase antibody (1:100, Abcam), or alternatively, with a rabbit anti-histone H3 (citrulline R2+R8+R17) antibody (1:500, Abcam). Cells were further incubated with a secondary goat anti-rabbit Alexa Fluor 488-conjugated antibody (1:1,000, Cell Signaling Technology), counterstained with DAPI and mounted in Dako fluorescent mounting medium (Dako). NETs were finally visualized using an inverted microscope (Eclipse Ti-U 100, Nikon) and the NIS Elements BR 3.10 software package.