Qimaging camera

Identification of NETs in this study was similar to a previously published protocol.^{10 (link)} Briefly, 2 × 10⁵ BMDNs were seeded onto poly-l-lysine-coated coverslips (Sigma-Aldrich). In experiments without stimulation or with stimulation with 2% serum (extracted from pristane-treated WT or Mfge8^−/− mice for 6 months), the cells were incubated for 2 h. In experiments with 1 μg/ml LPS, 100 nM PMA or 100 ng/ml MIP-2 stimulation, the incubation was for 4 h. NETs were stained with rabbit anti-histone H3 (citrulline R2+R8+R17) antibody and mouse anti-MPO antibody, followed by incubation with Alexa Fluor 647-conjugated mouse IgG and Alexa Fluor 488-conjugated rabbit IgG (Abcam). DNA was stained with DAPI. Images were collected with Olympus BX51 microscope and Qimaging camera, typically at original × 400 magnification.

Lung, spleen and liver tissues were fixed with formalin, embedded in paraffin and sectioned. Then, the tissue sections were stained with H&E or Masson's-trichrome stain. The prevalence of lungs with hemorrhage was estimated and the score of the left lobe was calculated using H&E staining: 4, 75–100% 3, 50–75% 2, 25–50% 1, 0–25% and 0, no hemorrhage.^{23 (link)} For the in situ TUNEL assay, cell death was determined using a TUNEL staining kit (Roche Diagnostics, Indianapolis, IN, USA).^{21 (link)} Briefly, sections of paraffin-embedded lung tissues were deparaffinized and subjected to antigen retrieval. Then, the sections were incubated with a mixture of TdT and fluorescence-labeled nucleotides. The prepared sections were examined with Olympus BX51 microscope (Olympus, Tokyo, Japan) and Qimaging camera (RoHs, Surrey, British Columbia, Canada). For pulmonary immune complex deposition, the frozen sections were stained with FITC-conjugated anti-C3 and Alexa Fluor 647-conjugated anti-mouse IgG (Abcam, Cambridge, UK). DNA was visualized using DAPI (Sigma-Aldrich), and the sections were examined with Olympus BX51 microscope and Qimaging camera.