Leukocyte Migration In Vivo Figure 7. Morphological changes and polarization of interstitially migrating leukocytes. Freeze-frames from in vivo microscopy video recordings obtained 60 min after microinjection of PAF (A), 60 min intrascrotal injection of PAF (B), and 60 min microinjection of PAF in combination with superfusion of the Rho kinase inhibitor Y27632 for 30 min (C; objective magnification 406). A: directional locomotion of a leukocyte towards the attractant with protrusions of lamellipodia at the front and a trailing uropod after microinjection of PAF. B: polarized leukocyte during random migration in the interstitial tissue after intrascrotal injection of PAF. C: blocking effect of Rho kinase inhibitor Y27632 on leukocyte polarization induced by microinjection of PAF. D: leukocyte polarization was measured as cell eccentricity: long axis (shown in black line)/short axis (depicted as interrupted line). Quantitative data of cell eccentricity after microinjection of saline, MIP-1a, PAF, E. coli, intrascrotal injection of PAF, as well as after microinjection of PAF with superfusion of Y27632; mean6SEM; *p,0.05 vs. saline, # p,0.05 vs. Y27632; n = 15. doi:10.1371/journal.pone.0004693.g007 establish a chemotactic gradient and mimic the local release of mediators during inflammation. Thus, at begin of the study, we evaluated how a microinjected chemoattractant would be distributed in the tissue after microinjection. For this, the distribution of fluorescent dye rhodamine 6G was analyzed after its microinjection. Analysis of fluorescence intensity showed that the concentration of rhodamine 6G was higher on the ipsilateral side up to 60 min after microinjection. However, it is worth to be noted that the distribution of the fluorescent dye may be different from the tissue distribution of a chemoattractant. Furthermore, in additional experiments, microinjection of MIP-1a was performed at different distances from the venule: 25–50 mm, 75–100 mm, and 175–200 mm. We found that a distance of 25–50 mm was optimal for inducing leukocyte migration since microinjections at 75– 100 mm and 175–200 mm initiated less leukocyte adhesion and transmigration. Taken together, these observations suggest that microinjection of MIP-1a generates a stable source of chemoat- tractant with slow diffusion within the interstitial tissue of the cremaster muscle. In a next set of experiments, we applied different chemoat- tractants (MIP-1a and PAF) and bacteria (E. coli) via microinjec- tion and analyzed leukocyte adhesion, transmigration and interstitial migration. First, we found that microinjection of MIP- 1a, PAF, or E. coli induced a significant increase in leukocyte PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4693