Platelets express higher levels of IL-1α compared with IL-1β. IL-1α and IL-1β levels in platelet lysates from WT or IL-1α/β^−/− mice (A), or rats (B) were quantified by ELISA immediately after isolation. Lysates of rat platelets (lanes 1 and 2) or recombinant rat IL-1α (rIL-1α) were analyzed by Western blot for IL-1α (C). Flow cytometric dot blots of 2-color fluorescence staining of rat platelets with anti–IL-1α (or IgG1) followed by anti–mouse allophycocyanin-conjugated antibody and anti–CD61-FITC (D). Immunostaining for flow cytometry was performed on nonpermeabilized (D top) or permeabilized (D bottom) rat platelets, and numbers in corners indicate percentage of population in each quadrant (D). Platelets were immunostained with anti–IL-1α antibody, without (Ei,Eii) or with (Eiii) preadsorption of the antibody with recombinant IL-1α. Platelets were immunostained with anti-GPV antibody (Ev), which localized to regions of IL-1α staining (Evi, merge of Eii and Ev). Isotype control antibodies for anti–IL-1α (IgG1) or anti-GPV (hamster IgG3) did not stain platelets (Eiv). Scale bars represent 5 μm. Images were acquired on a Delta Vision RT restoration microscope using a 100×/1.4 Plan Apo objective, 1.6× auxiliary magnification, and the Sedat filter set (E). The images were collected using a CoolSNAP HQ (Photometrics) camera with a Z optical spacing of 0.2 μm, raw images were deconvolved using the softWoRx software, and maximum intensity projections of these deconvolved images are shown (E). A, n = 3; B, from left to right n = 6, 7. ND indicates not detected. Data are mean ± SEM and representative of at least 3 independent cultures.