Araldite

In accordance with previously described methods (Beneduci et al., 2005), hippocampal neuronal suspensions were centrifuged and pellets fixed in 3% glutaraldehyde solution (pH 7.3) for approximately 2 hours at 4°C. Cell suspensions were rinsed twice with PBS, post-fixed in 1% osmium tetraoxide solution (pH 7.3), and incubated for 2 hours at 4°C. Cells were dehydrated in a graded alcohol series for 5 minutes each incubation. Dehydrated pellets were embedded three times with propylene oxide for 1 hour each time, infiltrated with a resin/propylene oxide mixture (1:1 ratio) for 2 hours, and then resin only for 12 hours at ambient temperature. The inclusion was made with Epon 812 and Araldite (Electron Microscopy Sciences, Hatfield, PA, USA), and polymerization performed at 60°C for 48 hours. Sections were cut parallel to the culture substrate at a thickness of 70 nm, and collected on 300-mesh copper grids. Ultrathin sections were stained with uranyl acetate and lead citrate counterstained, before examination with an EM900 Zeiss transmission electron microscope (Zeiss, Oberkochen, Germany).

Fresh sputum plugs were fixed in a mixture of 4% formaldehyde and 2% glutaraldehyde, omitting the DTT treatment, and embedded in Araldite (Electron Microscopy Sciences, Hatfield, PA, USA) for ultrastructural analysis. Thin sections were then mounted on nickel grids for observation in a Zeiss LEO 906 E Electron Microscope (Carl Zeiss, Oberkochen, Germany). Some samples were also embedded in LR-White (Electron Microscopy Science), a hydrophilic resin, in order to perform DAPI staining to evaluate the extracellular DNA traps.