Sp8 inverse confocal microscope

Adult mice were sedated with isoflurane, perfused and sacrificed. Coronal brain slices with 60 µm diameter were obtained by cryotome sectioning and subsequently permeabilised with 2% Triton X-100 in phosphate-buffered saline (PBS) for 24 h. Slices were incubated with primary antibodies for 48 h in PBS with 0.1% Tween-20 (PBS-T). After three washes with PBS-T, slices were incubated with species-cross-absorbed secondary antibodies in PBS-T for a further 48 h. As preparation for tissue clearing, slices were fixed after three washes with PBS in 4% formaldehyde (weight per volume) in PBS for 1 h at 4 °C. Tissue clearance was performed using ScaleA2 (4 M urea, 10% (weight per volume) glycerol, 0.1% (weight per volume) Triton X-100 and 0.1× PBS) for 3 days, as described previously^{37 (link)}. Transparent slices were mounted onto glass slides using Mowiol with 4 M urea. Slices were analysed using a Leica Sp8 inverse confocal microscope equipped with a ×63 objective (NA 1.7), whereas the channels were separately acquired by a sequential scanning mode. The correlation between the intensity of DBN and the intensity of pS647-DBN was analysed by tracing four random lines across the image and performing a plot profile to obtain the fluorescent intensity at each pixel.

10⁵ isolated human or mouse neutrophils were seeded on glass coverslips in tissue-culture plates and incubated with PMA (100 ng/ml, Sigma-Aldrich), Ionomycin (1 µM, Sigma-Aldrich), or 5 × 10³C. albicans preformed hyphae for 2.5 h at 37°C. Cells were then fixed with 4% paraformaldehyde, permeabilized with 0.01% TritonX-100 for 10 min, and blocked with 5% donkey serum for 30 min at room temperature. Cells were stained with goat polyclonal anti-MPO (R&D systems), rabbit polyclonal anti-CitH3 (Abcam), and combined rat anti-S100A8 and anti-S100A9 (clone 10 and clone 46 respectively, kindly provided by C. Urban, Umeå, Sweden). Secondary antibodies were donkey anti-goat IgG (Jackson ImmunoResearch), goat anti-rat IgG (Abcam), and goat anti-rabbit IgG (Jackson ImmunoResearch). The PKH26 Red Fluorescent Cell Linker Kit (Sigma-Aldrich) was used to stain lipids of the neutrophil plasma membranes. DNA was stained with 4′,6′-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) and NETs were visualized using a Leica SP8 inverse confocal microscope and analyzed with FIJI software.

Longitudinally cut tongue halves were placed into 1.5 ml safe-lock tubes containing 800 ml sterile 20% KOH solution and incubated at 60°C and 300 rpm for at least 30 minutes. Tubes were inverted regularly to prevent sedimentation of tissue fragments. After complete dissolution of the tissue, the suspension was centrifuged for 10 minutes at 6’000 rpm and supernatant was discarded. C. albicans cells were suspended in 495 μl Tris-HCl (0.1 M, pH 9) and 5 μl calcofluor white (5-10mg/ml in 0.1 M Tris-HCl, pH 9) to stain the cell wall. After 10 minutes of incubation in the dark, C. albicans cells were sedimented for 10 minutes at 6’000 rpm and washed twice with 1 ml Tris-HCl (0.1 M, pH 9). Fungal cell pellets were then resuspended in 20 μl PBS and images with a Leica SP8 inverse confocal microscope.