Figure 3. Mice exposed to lewisite and PAO on skin display NETosis in the lung.
(A) dsDNA in the BALF of control and lewisite groups (n = 5/group). (B) Immunofluorescence staining of lung sections and (C) measurement of fluorescence intensity for Cit-H3 (red) and NE (green) from control (n = 5) and lewisite groups (n = 5). Scale bar: 100 μm. (D) IHC for PAD4 in lung sections from control and lewisite groups (n = 5/group). Scale bar: 100 μm. Inset: original magnification, ×200. (E) Measurements of DAB intensity for PAD4 from IHC images. An average measurement from 5 mice was calculated from the average of 8 images. Mice were subjected to skin exposure to PAO and sacrificed after 6 hours (F–L). (F) BALF cell pellets stained with hematoxylin (nuclear stain). Scale bar: 5 μm. (G) Percentage of neutrophils counted in BAL cell pellets (n = 5/group). (H) Measurement of dsDNA in BALF (n = 5/group). (I) IHC images for PAD4 staining (n = 5/group). Scale bar: 80 μm. The inset demonstrates an enlarged area of the image: original magnification, ×200. (J) Measurements of DAB intensity from IHC for PAD4. An average measurement from 5 mice was calculated from the average of 8 images. (K) IHC of the lung sections for Cit-H3. Scale bar: 100 μm. (L) Mean intensity of DAB for PAD4 from IHC images. An average measurement from 5 mice was calculated from the average of 8 images. All data are representations of one experiment out of 3 independent experiments. Each dot represents an individual mouse in the graphs. Data are shown as the mean ± SEM. **P < 0.01. Statistics: 2-tailed t test.