Fig. 2

Cosnfocal microscope imaging of NETosis in EC specimens. A, E Two representative samples (grade G2 and G3) stained with anti-NE and anti-histone H2B antibodies. The yellow color on the images indicates the colocalization between the two markers. B, F Stainings of the same samples as A and E with anti-NE antibody and DAPI. The white color on the images indicates the colocalization between NE and DNA; when in the nucleus, colocalization strongly suggests neutrophils activation. 40 × oil objective, confocal single stack. C, G 3D reconstruction of magnifications of the areas inside the white squares in B and F respectively. Merge of 3 fluorochromes are shown. D Single staining of panel C detail highlights extracellular extrusion of DNA and citH3. 60 × oil objective. H, M Stainings of the same samples as A and E respectively with anti-citH3 antibody and DAPI. 40 × oil objective, confocal single stack. I, L, N, O Magnifications of the areas inside white squares in H and M respectively. In L and O the white color indicates colocalization between citH3 and DNA in the nucleus, cytoplasm and extracellular space. 60 × oil objective. Thin white arrows indicate the areas of colocalization and extracellular extrusion of NE, DNA and citH3. All scale bars indicate 50 µm. P Schematic representation (left) flanked by IF images taken from EC tissues (right), of the various stages of NETosis. Panels I, II, III show resting neutrophils, with trilobed nucleus and intact cytoplasm containing granules of NE. In IV, V, VI the nucleus has lost its classic trilobed shape, a yellow color is observed due to the nuclear co-localization of the two markers, testifying the transfer of NE into the nucleus. Panels VII, VIII and IX show the release of neutrophil extracellular traps (NETs). All scale bars indicate 5 µm