Dmr upright fluorescence microscope

Freshly harvested stem segments were fixed in 70% (v/v) ethanol and cross-sectioned to approximately 120 μm. Sections were stained with toluidine blue for 1 min and washed twice with water. Sections were viewed and photographed under a Leica DMR upright fluorescence microscope using brightfield settings.

Neutrophils seeded on glass coverslips were stained and quantified as previously described (Brinkmann et al., 2012 (link)). Briefly, cells were fixed in 2% paraformaldehyde (PFA) post-NET induction, permeabilized on 0.5% Triton-X100, blocked for 30 min in blocking buffer. Neutrophils were then stained with the following primary antibodies: anti-neutrophil elastase (Calbiochem: 481001, RRID:AB_212213) and antibodies directed against the subnucleosomal complex of Histone 2A, Histone 2B, and chromatin ([Losman et al., 1992 (link)], generated in house). The secondary antibodies donkey anti-mouse Cy3 (Jackson ImmunoResearch Labs: 715-175-150, RRID:AB_2340819) and donkey anti-rabbit Alexa Fluor488 (Life Technologies: A11008, RRID:AB_143165) were used. Finally, the samples were stained with Hoechst 33342 (Immunochemistry: 639, RRID:AB_2651135) and mounted with Mowiol. Image acquisition was c using a Leica DMR upright fluorescence microscope equipped with a Jenoptic B/W digital microscope camera and analysed using ImageJ/FIJI software.

At the end of each imaging session, or 3 days after the fluorescent retrobeads injection, the mouse was overdosed (100 mg/kg ketamine and 0.14 mg/kg medetomidine, i.p.) and perfused transcardially, first with PBS and then with 4% paraformaldehyde in PBS. After 2 days in fixative, and one in 20% sucrose solution, the brain was sectioned in the coronal plane at a thickness of 60 μm on a freezing stage microtome and mounted on glass slides. The sections were examined with a Leica DMR upright fluorescence microscope. Excitation wavelength was 480 nm for visualizing eGFP and 530 nm for rhodamine retrobeads. Images were processed offline using ImageJ (NIH).