Leukocyte Migration In Vivo Figure 3. Leukocyte adhesion and transmigration. Numbers of adherent (A) and transmigrated (B) leukocytes at 60 minutes after microinjection of saline, MIP-1a, PAF, or E. coli, and after intrascrotal injection of PAF (PAF i.s.) were counted on the vessel side ipsilateral to the microinjection site (black bars) and on the contralateral side (white bars); mean6SEM; n = 6; *,0.05 vs. ipsilateral side, #,0.05 vs. PAF- contralateral side; E. coli n = 3; PAF i.s. n = 3. C–F: intravital microscopic images from the murine cremaster muscle demonstrate adherent and emigrated leukocytes (arrows) after microinjection (C and E, respectively) and intrascrotal injection of PAF (D and F, respectively); asterisk shows the site of microinjection; objective magnification 206; scale bar 25 mm. doi:10.1371/journal.pone.0004693.g003 1.8 as compared to those after microinjection of saline (Fig. 7A, D). Although the intrascrotal injection of PAF also induced leukocyte polarization, the value of cell eccentricity was significantly less as compared with that after microinjection of PAF (Fig. 7B, D). Application of Y27632 abolished the PAF-induced leukocyte polarization, and the ratio between cell long and short axis was less than 1.2 (Fig. 7C, D). Migration patterns of different leukocyte subsets In the final part of the study, we used a combination of RLOT and in vivo fluorescence microscopy in Cx3CR1gfp/gfp mice in order to analyze the migratory behavior of GFP-positive cells (mono- cytes) as well as GFP-negative cells (neutrophils) after microinjec- tion of MCP-1. In our experiments, the number of transmigrated monocytes and neutrophils was significantly increased at 60 min after microinjection of MCP-1 (Fig. 8) as compared to baseline conditions (data not shown). Transmigration of leukocytes had a directional character as shown by the distribution of the extravasated monocytes and neutrophils predominantly on the vessel side ipsilateral to microinjection (Fig. 8A). The quantita- tive analysis of leukocyte interstitial migration demonstrated that perivenular microinjection of MCP-1 induced target-oriented migration of neutrophils within the interstitial tissue as shown by increased curve-line and straight-line migration velocity and directionality (Fig. 8A–D) compared to those after microinjection of saline (Fig. 5A, B, C). Curve-line velocity of neutrophils was significantly reduced at 70 and 80 min as compared to that at PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4693