High-resolution 3D imaging and topological mapping of the lymph node conduit system
Three-dimensional EVIS images of a popliteal LN with WGA-labeled conduit paths contain brightly labeled multilobular spaces (red arrowheads) within the otherwise unstained B cell Fo underneath the SCS that can be repeatedly seen in 3D projections of 20 μm thickness (A) and 2D image slices (B). In immuno-labeled tissue sections of WGA-perfused inguinal LNs, WGA (green) is found in follicular conduits descending from the SCS (white arrowheads) connecting to cellular clusters expressing the FDC marker CD21/CD35 (C, D). The morphology and location of WGA-labeled cell clusters within B cell follicles (D) are generally consistent with the anatomy of FDCs in these regions (E, F) as co-staining with markers for B cells (CD45R/B220), T cells (CD3), and collagen I confirms. High-resolution confocal images (with a voxel resolution of 0.36 × 0.36 × 1 μm) of a WGA-perfused popliteal LN provide insights into the staining pattern within and around the WGA accumulations (G) and show a particularly bright cluster in several z steps (H) directly neighboring spaces with weaker labeling (red arrowheads). Images are representative of at least 6 LNs (from N = 5 mice) in which multilobular cell clusters could be observed. See also S4 and S5 Videos. CD,; EVIS, extended-volume imaging system; FDC, follicular dendritic cell; Fo, follicle; LN, lymph node; SCS, subcapsular sinus; WGA, wheat germ agglutinin.