Intracellular event during saliva-induced NETosis. Neutrophils were left nontreated or were stimulated with saliva for 15 minutes and examined by immunofluorescence microscopy and electron microscopy. All fluorescence micrographs were acquired with a Zeiss LSM700 Axioimager M2 equipped with a 4-stack laser system (−405, −488, −555, −635 nm wavelength) using Zen (Zeiss) software, with a 63× oil immersion objective. Electron micrographs were acquired using a JEOL JEM 1230 transmission electron microscope and a JEOL JSM-350 scanning electron microscope with a secondary detector. (A) Immunofluorescence microscopy shows that staining for SUN2 found in the nuclear membrane is dramatically changed after 15 minutes stimulation by saliva. Scale bars represent 10 μm. (B) Transmission electron microscopy demonstrating that the nuclear membrane is lost after stimulation by saliva. Arrow heads indicate nuclear membrane. Scale bars, 50 nm. Larger images found in supplementary Figure 5. (C) Scanning electron microscopy of neutrophils demonstrating no visible changes in the plasma membrane after 15 minutes of saliva stimulation. Scale bars, 100 μm. Larger images found in supplementary Figure 4. (D) Confocal immunofluorescence microscopy for the cytosolic protein S100A8. Scale bars, 10 μm. (E) Confocal immunofluorescence microscopy and immunoelectron microscopy the azurophilic granule protein elastase (green or 5 nm gold particles) and CD63 (red or 10 nm gold particles) present in the azurophil granule membrane. Yellow color depicts colocalization. Many ribosomes of approximately the same size as the 10 nm gold particles are found around the granules in the electron micrograph. Arrow heads indicate elastase in the granule matrix and CD63 in the granule membrane. Scale bars, 10 μm for fluorescence micrographs and 100 nm for electron micrographs. (F) Confocal immunofluorescence microscopy and immunoelectron microscopy demonstrates the specific granule protein hCAP-18 (green or 5 nm gold particles) and CD18 (red or 10 nm gold particles) present in the membrane of specific granules. Yellow color depicts colocalization. Many ribosomes of approximately the same size as the 10 nm gold particles are found around the granules in the electron micrograph. Arrow heads indicate hCAP-18 in the granule matrix and CD18 in the granule membrane. Scale bars, 10 μm for fluorescence micrographs and 100 nm for electron micrographs.