Fig. 2

Induction of CitH3 in the brain tissue and PMNs following ischemic insult (A-D) CitH3 protein levels in the cortical penumbra and cortical core (defined in Fig. 1A) were measured by immunoblotting at 3, 6, 12, 24, 48, and 96 h after MCAO surgery. (E-G) CitH3 and MPO levels were assessed by immunoblotting in PMNs isolated at 12, 24, 48, and 96 h after MCAO surgery. (H) The amounts of cell-free dsDNA in the serum were assessed using Quant-iT PicoGreen dsDNA reagent. (B, D, F-H) Data are presented as the mean ± SEM (n = 4). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to sham-operated groups. (I-K) Coronal brain sections were prepared from sham controls (I) and the cortical penumbra or core of the ischemic hemisphere (J, K) at 48 (J) or 96 h (K) post-MCAO. Triple immunofluorescence staining was conducted with anti-Ly6G antibody, anti-PAD4 antibody, and DAPI (I, J) or with anti-CitH3 antibody, anti-MPO antibody, and DAPI (K). Arrows indicate co-localization of anti-PAD4 with anti-Ly6G or anti-CitH3 with anti-MPO in brain parenchyma, while double arrows indicate the co-localization of anti-PAD4 with anti-Ly6G or anti-CitH3 with anti-MPO within blood vessels. Arrowheads indicate the co-localization of anti-PAD4 with neuron-like cells. Scale bars represent 50 μm