Formation of the conduit system during LN organogenesis. (A) Immunofluorescence staining of E15, E16, P0 (40×/1.4 NA water lens), and P4 (25×/1.4 NA) iLN sections showing the development and organization of the ERTR7⁺ reticular network (red) and nuclear staining DAPI (blue). Note that the cells in the center of the anlagen at E15 were ERTR7⁻ and by E16 ERTR7⁺ structures were seen toward the center of the organ (arrowhead). (B) Changes in stromal-cell populations during iLN ontogeny. FACS analysis of single-cell suspensions of iLNs at the times indicated showing the recruitment of bone marrow–derived cells as an increased percentage of CD45⁺ cells and the concomitant changes in the CD45⁻ stromal cells, including the appearance of VCAM-1⁺ICAM-1⁺ mature stromal organizer cells and the progressive disappearance of VCAM-1⁺ICAM-1⁻ cells. Percentages in scatterplots correspond to CD45⁻ stromal cells according to their expression of VCAM-1 and ICAM-1. (C) FACS analysis of single-cell suspensions from E15, E16, and P0 iLNs. Percentages shown in scatterplots correspond to CD3⁻ cells according to their expression of CD45 and CD4. Note that LTic's are CD45⁺4⁺, and the largest population present is CD45⁺4⁻. Results are representative of 4 independent experiments.