Fig. 1. Transmigrated leukocytes express 6 integrins on their cell surface in IL-1ß-stimulated mouse cremaster muscles, as observed by confocal microscopy. Mouse cremaster muscles were injected i.s. with IL-1ß (30 ng) and, 4 hours later, the tissues were dissected away from the animals, fixed in 4% PF and incubated with the primary mAb against 6 integrins, GoH3. Binding of GoH3 was detected using a secondary antibody conjugated to Alexa Fluor 488. Samples were observed at RT using a Zeiss LSM 5 Pascal confocal laser-scanning microscope (using a 40x water-dipping achroplan objective with a numerical aperture of 0.75) equipped with an argon laser (excitation wavelength of 488 nm). Typically, multiple optical sections of tissue samples, running through the whole depth of the tissue, were captured and imaged using the software's automatic scanning mode. In selected experiments, z-axis stack images (collected at every 0.8-1.2 µm depth) were saved and used for three-dimensional reconstruction analysis using the LSM 5 Pascal software (version 3.2). The images show expression of 6 integrins on leukocytes in the extravascular tissue, displayed as fluorescence (left), differential-interference contrast (DIC) (middle) and merged (right) patterns. (A) This series was obtained from the mid-level section of the marked leukocyte (yellow arrow), demonstrating a pronounced ring-like expression profile, indicating cell-surface expression. (B) A three-dimensional reconstructed image of the same tissue shown in two dimensions in A, illustrating the more global-like distribution of 6 integrins on transmigrated leukocytes. Leukocytes and vessel walls are shown by yellow and red arrows, respectively. Scale bars, 10 µm.