Platelet-derived IL-1α drives neutrophil TEM. (A) The effects of CM from resting platelets or PMA-activated platelets (± IL-1RA) on neutrophil TEM. The effects of recombinant IL-1α (0.003-30 U/mL) on neutrophil TEM (B, light microscope images of migrated neutrophils in abluminal transwell compartment of MBECs in response to treatment with vehicle or 30 U/mL IL-1α, scale bar represents 40 μm; C, quantification). Brightfield images were collected on an Olympus CKX31 cell culture microscope (Olympus) equipped with a 40×/0.55 objective and a Motic 2300 digital camera and Motic imaging software (Motic; B). Immunodetection of SJC-positive neutrophils in, or in close proximity to (arrowheads), ICAM-1-positive brain vessels in mice exposed to cerebral ischemia (Di; and Dii, high magnification). CD41-positive platelets are shown in red (scale bars represent 20 μm). Fluorescent images were acquired on a Olympus BX51 microscope using a 40×/0.75 Plan Fln objective and FITC, Texas Red, and DAPI filter sets and captured using a CoolSNAP ES camera through MetaVue Software (Di) or on a Delta Vision RT restoration microscope using a 40×/1.3 Plan Apo objective and the Sedat filter set (Dii). The images were collected using a CoolSNAP HQ camera with a Z optical spacing of 0.2[ mu]m, raw images were deconvolved using softWoRx software, and maximum intensity projections of these deconvolved images are shown (Dii). A, n = 3; C, from left to right, n = 6, 7, 5. Data are mean ± SEM. (A) ***P < .001, **P < .01, vs vehicle. (C) **P < .01 vs vehicle.