A and B: Gross structure of splenic longitudinal sections showing IL-4 production (blue) and CD3 positive cells (red) at 40X magnification. A, WT 8-week section showing red-stained T cell zones and non-detectable IL-4. B, 8-week section of Ttc7fsn spleen divided into 4 parts showing dramatic enlargement of organ size and disintegration of distinct T cell zones. IL-4 expression was detectable throughout spleen, but especially in follicular areas as indicated by arrows. 40X magnification. C and D: IL-4 expression in 8-week spleen visualized with laser scanning confocal microscopy using a 20x objective. Spleen sections were stained for IL-4 (green), CD3+ T cells (red) and IgM+ B cells (blue). Ttc7fsnspleen (D) shows an increase in IL-4-positive intensely stained T cells (yellow) as compared to WT littermate (C). In addition, T cells are located outside of the follicle in the B-cell-rich zone consistent with the location of pStat6 positive B cells (figure 3). All staining was repeated three times in three separate animals with similar results.
These are some figures from my lab examining B cells from the Autoimmune prone flaky skin mutant mice.
Purified splenic B cells were unstimulated (0) or stimulated with IL-4 for 15 and 30 minutes, then stained for Stat6 (red) and the nucleus (green) and visualized with confocal microscopy using a 60x objective. Co-localization of Stat6 with the nucleus, denoting activation, is yellow. Ttc7fsn B cells show more yellow, activated cells than normal even without stimulation, suggesting pre-activation in vivo.