B10.BR mice were immunized with 10nmole HEL (A, C–E) or Hilyte Fluor 555-labeled HEL (B) in complete Freund's adjuvant. Popliteal lymph nodes were harvested at the indicated time. (A, D) 5μm frozen sections were stained with anti-CD3 (red) and anti-Gr-1(green) for epifluorescent microscopy (A); anti-CD11c (blue) and anti-Gr-1 (green) for confocal microscopy (D). Shown are representative from 8 individual experiments using 2 mice for each time point. Histological analysis disclosed the presence of typical neutrophils as described above. (C) Flow cytometric analysis of CD11b+ Ly-6G+ cells in popliteal lymph nodes at indicated time points was done after 10nmole HEL immunization. Data is a representative result taken from four experiments. Statistical analysis was processed by Mann-Whitney test. (E) LysM-eGFP/CD11c-eYFP mice were immunized with 10nmole HEL/IFA in the footpad. Popliteal lymph nodes were removed for two-photon microscopy. Contacts between DC (green) and neutrophils (blue) were visualized in the cortical sinus at 3 hours post-immunization. Scale bars: 200μm (A, B); 30μm (D); 150μm (E, left); 30μm (E, right).