Figure 4.

Formations of germinal centers (GCs) in the spleen are dependent on in vivo MyD88 signaling in macrophages upon immunization with toll-like receptor ligand-based adjuvants. (A) GC responses examined by confocal microscopy 7 days after the second immunization, 8 μm thick spleen sections were prepared and examined as described “Animals and Methods.” Sections were stained for IgD follicular B cell marker (red), GC B cell marker GL7 (green), and T cell marker TCR-β (cyan). (B) A spleen section with the GC area measured using the freehand selection tool in ImageJ, here the area of GC (green) is encircled in white. (C) Quantitation of GC areas by ImageJ. There were three mice per immunization group, 6 sections per spleen that are at least 40 μm apart and a total of 18 sections were examined per group. (D) Percentage of GC cells from spleen was measured by flow cytometry 7 days after the second immunization and gating for B220⁺CD4⁻CD19⁺GL7⁺GC cells. The results shown are representative of at least two experiments with a total of eight mice per immunization group for the flow cytometry analysis experiment. One-way ANOVA with Tukey’s test were used (ns, not significant P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).