Analysis of in vivo dendritic cell migration from the skin via the lymphatic vessels to the lymph node. (A) Wild-type and Cdc42^−/− DCs were labeled with TAMRA (red) or Oregon Green 488 (green), and a 1:1 mixture was injected subcutaneously into the hind footpads of C57BL/6 mice and arrival in the popliteal lymph node (LN) was analyzed 48 hours later. (B) Immunofluorescence microscopy (Axio Imager; Zeiss) of 12-μm-thick LN cryosections. Counterstaining against laminin (Cy5, blue) distinguishes B-cell follicles (B) and T-cell cortex (T). Scale bar represents 200 μm. Objective: EC Plan-NEOFLUAR 10×/0.3 (Zeiss). (C) Quantification of immunofluorescence analysis. Three different layers of the T-cell cortex of each LN were documented and quantified by morphometric analysis (MetaMorph). Dotted line at 0.5: 50% of DCs that migrated into the LN are Cdc42^−/− DCs. Circles indicate single experiments (1 LN).