Localization of CR-Fc binding cells in lymph nodes with time after a primary immunization. Three-color confocal analysis was used in order to determine when and where CR-Fc binding cells appeared in the tissue. Cryostat sections prepared from murine lymph nodes obtained at days 0–4 (A–E) of a primary response were processed for IgM expression (red), N418 labeling (green), and CR-Fc binding (blue). The images were then digitally transformed using a computer program in order to map the MFI (in arbitrary units) for either single (CR-Fc+; N418+) or double (CR-Fc+/ N418+) positive cells. The resulting 3D immunohistograms revealed that CR-Fc binding cells accumulated with time in the outer part of the B cell follicles. Many of these cells were also N418+ (see the CR-Fc+/N418+ column). The scale 0–250 represents the MFI, with the colors yellow, blue, green, and red representing values in the 100–150, 150–200, 200–250, and >250 range, respectively.
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