PLT depletion reduces apoptosis in a murine stroke model in vivo. (A-E) Stroke was induced in C57BL/6 mice by tMCAO for 60 minutes. (A-B) Ischemic brain tissues were co-stained for PLT GPIbα (red), neuronal cells (NeuN, blue), and endothelial cells (CD31, green). Nuclei were stained using DAPI (displayed in white using Leica Confocal software version 2.61). Scale bar, 20 µm. (A) Shows representative images. (B) For quantification, PLTs outside of vessels were counted in a blinded fashion using fluorescence microscopy in healthy and ischemic brain tissue. Data are mean ± SEM and show the percentage of total PLTs per section (n = 6, 3 non-consecutive sections per animal were analyzed). (C) Identification of early (Annexin V⁺, PI⁻) and late (Annexin V⁺, PI⁺) apoptosis in CD45.2⁻ nonleukocytes that complex with CD41⁺ PLTs in stroke tissue and the contralateral control hemisphere (upper panel). Percentage of CD41⁺ PLT complexes with apoptotic nonleukocytes in the stroke hemisphere relative to the corresponding contralateral hemisphere was calculated (n = 4). Results are presented as mean ± SEM, *P < .05 (left, lower panel). FMO staining controls document staining specificity and gating strategy (right, lower panel). (D-E) Prior to stroke induction, animals were treated with either control serum (ctrl) or PLT-depleting serum (PLT depletion) resulting in more than 90% of PLT depletion (supplemental Figure 1). (D) Shows representative images of stained tissue sections from a control serum or PLT-depleting serum-treated mouse after induction of stroke. Nuclei were stained with DAPI (blue) and TUNEL-positive cells are depicted in red. Scale bar, 100 µM. (E) Quantification of apoptotic cells upon injection of control serum or PLT-depleting serum. For quantification, apoptotic cells were counted in a blinded fashion using fluorescence microscopy. Data are mean ± SEM and show the percentage of TUNEL-positive cells of total cell number per section (n = 6). *P < .05 vs control treated animals. FMO, fluorescence minus one.