CELLULAR INTERNET IN THE GERMINAL CENTER 117 MATERIALS AND METHODS Immunohistochemistry Human tonsils surgically removed from children with recurrent tonsillitis were used for both immunohis- tochemistry and culture (see later). Frozen sections 8 /xm thick were fixed in 4% paraformaldehyde, then in acetone for 5 min each, and allowed to dry for 2 hr. For single-antigen detection, the sections were treated for 2 hr with rabbit anti-Cx43 (HJ; Green and Severs, 1993), desmoplakin (Dpl2; gift from A.I. Magee, National Institute for Medical Research, Mill Hill, London), Ki-67, IgM, and IgD antibodies, or with mouse anti-Cx43 (Zymed, San Francisco; clone: CX- 1B1), CD21, and CD35 antibodies, respectively. The specific binding sites were revealed with either biotinylated goat anti-rabbit Ig-s, or anti-mouse Ig-s, followed by the use of a FITC-conjugated Streptavi- din reagent, for 1 hr each. For simultaneous antigen detection, pairs of rabbit and mouse antibodies were applied in mixtures for 2 hr followed by a mixture of TRITC-conjugated swine anti-rabbit Ig-s and FITC- conjugated goat anti-mouse Ig (Fab’2) for 1 hr. All antibodies were from DAKO (Glostrup, Denmark), except where otherwise noted. FDC-B-Cell Cultures The tonsils were minced and low-density FDC-rich germinal center B-cell fractions were separated as described by Lindhout et al. (1993). Briefly, tissue pieces were first digested in a mixture of Collagenase IV and DNase in Iscove’s modified Dulbecco’s medium. From the supernatants, cell fractions were separated on a BSA gradient (cells between the interfaces of 2.5% and 5% BSA were used) followed by Percoll gradient centrifugation. The cells of <1060 mg/ml density were cultured on 22 24-mm coverslips in IMDM supplemented with 0.1% genta- mycin and 10% fetal calf serum. Cell cultures were fixed onto the coverslips and immunostained in the same ways as the frozen sections (see earlier) at any time points of the investigation between 2 to 24 hr. Immunostaining for immunoglobulins and Cx43 was carried out in freshly separated, low-density cell fractions too. Acknowledgements We are grateful to Ernst Lindhout and Maike van Dartel for help in cell culture work and for their valuable comments in the interpretation of the results. We also thank The Wellcome Trust in England and the OMFB-British Council GB-26/95 and OTKA T022277 in Hungary for supporting this work. References Alves L.A., Campos de Carvalho A.C., Cirne Lima E.O., Rocha e Souza C.M., Dardenne M., Spray D.C., and Savino W. (1995). Functional gap junctions in thymic epithelial cells are formed by connexin 43. Eur. J. Immunol. 25: 431-437. Bruzzone R., White T.W., and Goodenough D.A. (1996). The cellular internet: On-line with connexins. BioEssays 18: 709-718. Clark E.A., Grabstein K.H., and Shu G.L. (1992). Cultured human follicular dendritic cells. Growth characteristics and interactions with B lymphocytes. J. Immunol. 148: 3327-3335. Clark E.A. and Ledbetter J.A. (1994). How B and T cells talk to each other. Nature 367: 425-428. Green C.R., and Severs N.J. (1993). Distribution and role of gap junctions in normal myocardium and human ischaemic heart disease. Histochemistry 99: 105-120. Granot I., and Dekel N. (1994). Phosphorylation and expression of connexin-43 ovarian gap junction protein are regulated by luteinizing hormone. J. Biol. Chem. 269: 30502-30509. Hossain M.Z., Wilkens L.R., Metha P.P., Loewenstein W.R., and Bertram J.S. (1989). Enhancement of gap junctional communi- cation by retinoids correlates with their ability to inhibit neoplastic transformation. Carcinogenesis 10: 1743-1748. Kim H-S., Zhang X., and Choi Y.S. (1994). Activation and proliferation of follicular dendritic cell-like cells by activated T lymphocytes. J. Immunol. 153: 2951-2961. Krenacs T., and Rosendaal M. (1995). Immunohistological detec- tion of gap junctions in human lymphoid tissue: Connexin43 in follicular dendritic and lymphoendothelial cells. J. Histochem. Cytochem. 43:1125-1137. Krenacs T., Rosendaal M., van Dartel M., and Lindhout E. (1997). Direct cell-cell communication in lymphoid follicles: Con- nexin43 gap junctions functionally couple follicular dendritic cells and follicular dendritic cells with B lymphocytes. Eur. J. Immunol. 27: 1489-1497. Kumar N.M., and Gilula N.B. (1996). The gap junction communi- cation channel. Cell 84: 381-388. Laird D.W., Puranam K.L., and Revel J.P. (1991). Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem. J. 273: 67-72. Lindhout E., Lakeman A., and de Groot C. (1995). Follicular dendritic cells inhibit apoptosis in human B lymphocytes by a rapid and irreversible blockade of preexisting endonuclease. J. Exp. Med. 181: 1985-1995.