Characterization of Early-Phase Neutrophil Extracellular Traps in Urinary Tract Infections
Fig 9
Detection of early-phase NETs by IF microscopy.
AUP aliquots were paraformaldehyde-fixed on glass slides on the day of specimen collection and stored at 4°C until further use. IF staining was performed with an MPO-specific polyclonal antibody followed by an anti-rabbit IgG conjugate to the dye CFl-555, and counterstaining with DAPI. Oil immersion microscopy (not confocal) was used for imaging with phase contrast and in blue and red channels. (a-d) sample #142; (e-h) sample #146; (i-l) sample #151; (m-p) sample #157. Sample #142 shows evidence of lobulated nuclei and no evidence of extracellular chromatin release (a), intact granular structures and well-defined cell perimeters (b), MPO staining in accordance with intact granules (c), and no co-localized MPO/chromatin staining (d). Sample #146 has intact neutrophils, but also some cells where nuclei fill the entire cell space (e) and granules are diminished in the perimeter of cells according to staining for MPO (g). Co-localization of nuclei and MPO is visible in the cell perimeter suggesting the emerging loss of nuclear membranes (h). Sample #151 shows less regularly shaped nuclei with fainter staining in their perimeters suggesting nuclear membrane disintegration (i), and patchy granular staining as described above (k); MPO and nuclear staining with DAPI overlap (l). Sample #157 shows areas of flattened and disintegrating cells (m, n) and streaks of extracellular DNA (m) that co-localizes with MPO staining (o, p). The closed white arrows point to cells with intact nuclei and well-distributed cytoplasmic granules. Open white arrows point to cells filled with chromatin and a patchy staining pattern for granules (MPO). Open yellow arrows point to disintegrated cells releasing chromatin from nuclei that co-localizes with MPO.