AID expression and class-switch recombination in secondary lymphoid organs and the thymus. AID expression was detected by conventional immunohistochemistry (A-C,E) and, in conjunction with CD20, by double immunofluorescent staining (D). AID is expressed in lymphoid follicles of the lymph node (A) and tonsil (C). In fully developed germinal centers (GCs), AID expression is predominately observed in the dark zone (C). Outside germinal centers, AID⁺ cells are found scattered throughout the T zone (A,C). The arrows in A indicate an extrafollicular area containing numerous AID⁺ cells. These AID-expressing cells are large and often display a distinct dendritic cytomorphology (B, and inset in B). Double staining of AID (red) and CD20 (green) shows coexpression of CD20 by AID⁺ cells (D). AID staining is largely limited to the cytoplasm (inset in B,D). Low numbers of AID⁺ cells are present in the thymic medulla (E) containing a Hassall corpuscle (arrow). AID⁺ asteroid cells of the thymic medulla morphologically resemble those in the T zone of the lymph node and tonsil (inset in E). (F) Quantification of AID mRNA by real-time PCR on microdissected tissue shows significant levels of AID expression in the germinal center, mantle zone (MZ), and T zone (TZ) of secondary lymphoid organs, and in the medulla (Me) of the thymus, but not in the thymic cortex (Co). Relative AID mRNA expression is given in arbitrary units as the mean (n = 3) and standard deviation. (G) Iγ-Cμ (1), Iα-Cμ (2), and Iα-Cγ (3) circle transcripts and GAPDH as a housekeeping gene were amplified by nested RT-PCR from microdissected tissue. Circle transcripts are detected in all 3 microcompartments of the lymph node and tonsil, and in the thymic medulla. No circle transcripts can be amplified from the cortical region of the thymus. Specific bands are indicated by arrowheads. The specificity of representative PCR products was confirmed by sequencing (GenBank accession nos. DQ083944-6). All data represent at least 2 independent stainings or measurements. Magnification: 10×/0.3 NA objective (A,C); 40×/0.75 NA objective (B); 63×/1.4 NA objective (D, and panels B and E's insets); and 20×/0.5 NA objective (E).