Stromal cells elicit formation of lymphoid-follicle–like structure with CD35⁺B220⁺ cells. (A) Immunostaining of lymphoid-follicle–like structures established by intradermal injection of PKH26-labeled CD35⁺B220⁺ cells (*) + splenic stromal cells (i), CD35⁺B220⁺ cells + brain-derived stromal cells (ii), or CD35⁺B220⁺ cells + TEL-2 stromal cells (iii) using mAbs for CD4, B220, and CD35. Nucleus was stained with DAPI. CD35⁺ reticulum in serial sections is marked by a yellow dotted line. Bars correspond to 200 μm. All images were photographed with an Olympus fluorescence microscope. (B) Lymphoid-follicle–like structures formed by CD35⁺B220⁺ cells together with brain stromal cells (i) or TEL-2 stromal cells (ii) were stained with anti-FDC-M1 mAb (green) and anti-B220 mAb (red). Lymphoid-follicle–like structures generated from B220⁺CD35⁺ cells and TEL-2 stromal cells were stained with FITC-conjugated anti-rat IgG and SA-Qdot605 as a negative control (iii). Representative of 3 independent experiments is shown with scale bar. Red bar, 200 μm; white bar, 15 μm. (C) Lymphoid-follicle–like structures generated by CD35⁺B220⁺ cells isolated from GFP-Tg mice and TEL-2 stromal cells were stained with anti-GFP mAb (green) and anti-FDC-M1 Ab (red). The serial section was stained with anti-GFP IgG conjugated with Alexa Fluor 488 and Cy3-conjugated antirat IgG as negative control. Representatives of 3 independent experiments are shown with scale bar. Green bar corresponds to 100 μm, purple bar corresponds to 30 μm, and white bar corresponds to 10 μm. Images were taken and analyzed with a DeltaVision RT system. Objective magnification, 20×/0.75 NA or 60×/oil 1.4 NA.