Extended Data Fig. 1: Antigens centralize on the FDC network.
a) Maximum intensity projection of confocal images of clarified LNs of mice immunized with IC-PE (magenta). FDC networks are in cyan (anti-CD21/35) (n = 6 LNs; 3 experiments). b) Image analysis to quantify antigen distribution within each B cell follicle. c) Confocal image of an FDC network (cyan) after immunization with two subsequent ICs in PBS analyzed 7 days after the first immunization (IC-PE; magenta) and 24 hours after the second (IC-488; yellow). Single-color images of IC-488 (left) and IC-PE (right) are shown below. Cyan line demarcates FDC network boundary based on anti-CD21/35 staining. Right, quantification of the distribution of both antigens on the FDC network. (n = 8 LNs; 2 experiments). d) Confocal image of an FDC network (cyan) after immunization with two subsequent ICs as in C for 14 and 7 days. Single-color images of IC-488 (left) and IC-PE (right) are shown below. Cyan line demarcates the FDC network boundary based on anti-CD21/35 staining. Right, quantification of the distribution of both antigens on the FDC network (n = 12 LNs; 2 experiments). e) Image of an LN FDC network (cyan) 56 days after immunization with IC-PE (magenta). Right, single-color image of IC-PE (gray) with cyan line demarcating the FDC network boundary based on anti-CD21/35 staining (n = 4 LNs; 1 experiment). f) Image of a draining LN 21 days after immunization with IC-PE (red). Naïve B cells are shown in gray (anti-IgD). (n = 4 LNs; 2 experiments). g) Image of a draining LN 7 days after immunization with AF555-labeled mi3-Spycatcher nanoparticles coated with YU-gp120-Spytag HIV envelope protein (magenta). FDC networks are shown in cyan (anti-CD21/35). White square indicates the region magnified. Cyan line demarcating the FDC network boundary based on anti-CD21/35 staining (n = 3 LNS; 2 experiments). h) Flow cytometry gating strategy to analyze FDCs. Quantitative data show the mean ± SD analysis by two-tailed t-test or one-way ANOVA with multiple comparisons.
