Figure 3. Detection of EGFP positive T lymphocytes in T zones of the spleen and lymph-nodes one month after thymic electroporation.

(A) Fluorescence analysis at 100X view of a splenic internal region reveals EGFP positive cells. (B) A nuclear counterstain (DAPI, blue) shows that these positive cells are localized in the white pulp. Insets represent a higher magnification at 400X view of the central arteriole area (ca) where a cluster of EGFP positive cells is observed. (C) 100X view of spleen immunohistochemistry analysis using an anti-EGFP antibody shows the distribution of transfected cells (arrows). (D) EGFP positive cells are localized near high endothelial venules (HEVs) stained with an anti-PNAd antibody (red) in lymph-nodes. (E) Sections of lymph-nodes and the spleen were analyzed by confocal microscopy after staining with a B cell specific antibody (B220) and an anti-CD3 antibody that specifically reacts with T lymphocytes. Multiple representative clusters of EGFP transfected cells were found mainly in T zones of different lymph-nodes and spleen sections. (F) Percentages of EGFP transfected CD3 positive T lymphocytes present within T zones of lymph-nodes and the spleen were calculated as described in methods. The mean percentage and the standard deviation of 3 individual organs (n = 3) were calculated. Abbreviations: wp = white pulp, rp = red pulp, ca = central arteriole, v = vessel.