Extended Data Fig. 2: The stromal network stretches upon lymph node swelling.

(a) 3D reconstruction of an entire homeostatic (day 0) popliteal lymph node (LN) by LSFM following clearing and staining of T-zone, follicles and lymphatic compartment volumes by CD3ε, B220 and LYVE-1 staining, respectively. LN surface and follicles are shown. Asterisks (*) indicate sites where follicles locally deform the LN’s capsule (left). Scale bar = 200 µm. 3D clipping of the dashed line encircled region (right). Follicle location with respect to capsule deformations are shown. (b) Example of T-zone deformation by B cell follicle (3D cropped). Asterisk (*) depicts the site at which underlying T-zone is indented by and curves around a follicle. Scale bar = 100 µm. (c) LN stromal network gap analysis. FRC-mGFP (top), segmented FRC network (middle), fitted circles in gaps (bottom), randomly colored, T-zone reticular cell (TRC) network in white. (d) Representative examples of the B cell follicle light zone (and for inflammation conditions also GC) FDC stromal network (within dashed line), and CXCL12⁺ reticular cell (CRC) stromal network (area between the dashed and solid lines) of LNs in homeostasis (day 0) and inflammation (day 4 and 14) from wild-type (WT) FRC-mGFP mice used for gap analysis as in c. Scale bars = 50 µm. (e) Representative examples of the CRC stromal network gap analysis as in d. (f) Averaged and smoothed distribution of the CRC stromal network gaps, plotted as the weighted area fraction as a function of the fitted circle diameter as in e (n = 5, 5, 5). (g) Quantification of the mean fitted circle diameter as in e (n = 5, 5, 5). Data from f, g, shown as mean. Datapoints in g represent independently measured follicles. Statistical analysis was performed using one-way ANOVA (g). All experiments were repeated independently (3 lymph nodes from ≥3 mice and ≥2 experiments). For statistical details, see Supplementary Information, table 1. ns, not significant.

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