Time-course analysis of all the GC structures and position of single B cells in intact LNs. (A) Mice were immunized with NP-OVA, and popliteal LNs were dissected at different time points and imaged by LSFM. Representative 3D-rendered images in volumetric and rainbow visualization modes are shown. Scale bar, 200 μm. (B–D) Quantification of GC number per LN (B), GC volume (C), and number of single cells per LN (D). (E) The number of total single cells per LN normalized to the total GC volume at different time points. Data for A–E were pooled from 3–7 independent experiments with a total of 4–23 LNs from 3–16 mice per group. *, P < 0.05; **, P < 0.01; ***, P < 0.0001; ns, not significant; one-way ANOVA with Tukey’s multiple comparisons test. (F) AID.Cre.tdTomato mice received GFP⁺ T cells and CFP⁺ B cells 12–14 d after immunization with NP-OVA. Representative 3D-rendered images of an LN and a single GC are shown. Scale bars, 200 μm (top panel); 100 μm (bottom panel). (G) Quantification of the number of individual tdTomato⁺ B cells in the T zone and in B cell follicles in single LNs after GC exclusion. Each pair of colored dots represents a single LN. **, P < 0.01; Wilcoxon matched-pairs signed rank two-tailed test. (H) The ratio of the number of individual tdTomato⁺ B cells in the B zone versus the T zone in individual LNs. Representative images and data for F–H were pooled from three independent experiments using a total of 10 LNs from eight mice. Unim., unimmunized.