Las x 3d version 3

For neutrophil extracellular trap (NET) detection, paraffin sections from animals at 4 dpi (serial cuts to HE and virus antigen staining) were analyzed. The immunofluorescence staining of paraffin section was performed as previously described.^{4 (link)}
Briefly, first mouse IgG2a anti-DNA/histone (MAB3864, Millipore; 0.55 mg; 1:500) and rabbit anti-human myeloperoxidase antibodies (A0398, Dako; 3.2 mg, 1:300) in blocking buffer were incubated overnight at 4°C. As secondary antibodies, goat anti-mouse antibody (Alexa488PLUS, Thermo Fisher Scientific) and goat anti-rabbit antibody (Alexa568, Thermo Fisher Scientific) were diluted 1:500 in blocking buffer. Nuclei were stained with Hoechst 33342 (Sigma). Samples were recorded using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with HCX PL APO 40 × 0.75–1.25 oil immersion objective. The settings were adjusted using isotype control antibodies in separate preparations. Representative three-dimensional (3D) images of z-stacks were constructed with LAS X 3D Version 3.1.0 software (Leica).

In order to further illustrate and confirm the localization of selected markers, confocal recordings of particular IF double labelling were generated. Laser scanning images were captured with a Leica TCS SP5 AOBS confocal inverted‐base fluorescence microscope (Leica Microsystems, Bensheim, Germany) with a HCX PL APO lambda blue 40x 1.25 oil immersion objective. For each double labelling, the laser settings were adjusted according to the appropriate controls. For the 3D images and movies, a series of optical sections (z‐stacks) were collected and analysed with LAS X 3D version 3.1.0 software from Leica. Z‐stack pictures were used and the background set to black by standard software settings.

Samples were recorded using a Leica TCS SP5 confocal inverted-base fluorescence microscope with a HCX PL APO 40 × 0.75–1.25 oil immersion objective. Settings were adjusted with control preparations using an isotype control antibody. For each sample 6–7 randomly selected images per independent experiment were acquired and used for quantification of the granulocyte number and the induced ET forming cells with an occurrence of a distinct extracellular off-shoot of the cells which is positive for histone-DNA complexes. For the 3D picture and movie, z-stacks were collected and analyzed with LAS X 3D Version 3.1.0 software from Leica. Fifty-six z-stack pictures were used and the background set to gray by standard software settings. The movie was made with the movie maker in the software.