Siglec-E is preferentially localized in areas of high CD11b staining on neutrophils spreading over fibrinogen. WT and siglec-E^−/− bone marrow cells were plated onto fibrinogen-coated coverslips for 20 minutes at 37°C. Neutrophils were fixed with 3.7% formaldehyde in phosphate-buffered saline and stained with polyclonal anti–siglec-E antibody conjugated to FluoProbe 547H and/or anti–CD11b-Alexa488 antibody. The cells were mounted in the presence of 4,6 diamidino-2-phenylindole to distinguish neutrophil nuclear morphology and were analyzed by confocal microscopy (LSM 700 microscope and Zen 2009 software; Carl Zeiss). Images were collected with an α-Plan-Apochromat × 100 NA 1.46 objective. Scale bars represent 5 µm. Image analysis was performed using Volocity software. The number of siglec-E foci per cell was quantified in areas of high CD11b staining compared with areas of low CD11b staining. High CD11b intensity was set to 1 to 3.3 standard deviations from the mean intensity, and low CD11b intensity was set to 0 to 1 standard deviations. The number of siglec-E foci was measured automatically in each region. Data are expressed as mean ± SEM from 10 images from 2 independent experiments containing ≥4 cells per image; *P < .05, paired t test.