Immunohistochemical labeling of CD35⁺B220⁺ cells with ICs. (A-C) CD35⁺B220⁺ cells retain ICs on their surface. CD35⁺B220⁺ cells (A) or CD4⁺ T cells (B) were labeled with ICs and the retained ICs (red) on cell surface were visualized with HRP-conjugated anti-rabbit IgG followed by GenPoint (Dako Japan) signal enhancement system and streptavidin-conjugated Qdot605. (C) Retention of ICs (green) on the surface of PKH26-labeled CD35⁺B220⁺ cells (red) in vivo was assessed by injecting IC-pulsed, PKH-26-labeled CD35⁺B220⁺ cells into naive C57BL/6J-Jcl mice. On day 8, the splenic sections were prepared and ICs (green) were stained with HRP-conjugated antirabbit IgG followed by GenPoint signal enhancement system and SA-488. ICs on the surface of CD35⁺B220⁺ cells are indicated by arrows. (D-F) CD35⁺B220⁺-cell–derived reticular cells retain ICs. CD35⁺B220⁺ cells isolated from GFP-Tg mice were injected intravenously into naive C57BL/6J-Jcl mice, and frozen sections of spleen were prepared 15 days later. Splenic sections were then incubated with ICs and freshly prepared mouse serum, and ICs (panels D and E, red) were visualized with goat anti-HRP Ab followed by Cy3-conjugated anti-goat IgG. CD35 (panel D, blue) or CD68 (panel E, blue) was visualized with biotin-conjugated anti-CD35 mAb or biotin-conjugated anti-CD68 mAb, respectively, followed by streptavidin-conjugated Cy5, and the GFP signal was enhanced with Alexa-488-conjugated anti-GFP antibody. CD68⁺ cells are indicated by arrows (panel E). In panel F, lymphoid-follicle–like structures were formed by intradermally injecting FDC-M1⁻CD35⁺B220⁺ cells from GFP-Tg mice together with TEL-2 stromal cells. A frozen section of lymphoid-follicle–like structure was labeled with ICs (red) and anti-CD35 mAb (blue), as described. All immunofluorescence images were obtained using a DeltaVision RT system. Red bars, 200 μm; white bars, 20 μm; green bars, 5 μm. Objective magnification, 20×/0.75 NA or 60×/oil 1.4 NA. A representative of 3 independent experiments is shown.