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).
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).
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