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Immunohistological and Flow Cytometric Analysis of Glycosphingolipid Expression in Mouse Lymphoid Tissues
Natasa Kovacica, Johannes Müthingb, and Ana Marusicaa Croatian Institute for Brain Research and Department of Anatomy, Zagreb University School of Medicine, Zagreb, Croatia
b Institute of Cell Culture Technology, Technical Faculty, University of Bielefeld, Bielefeld, Germany
Correspondence to: Natasa Kovacic, Croatian Inst. for Brain Research, Zagreb University School of Medicine, Salata 12, 10000 Zagreb, Croatia. E-mail: natasa@mef.hr
Expression of neutral glycosphingolipids (GSLs) and gangliosides in normal lymphoid tissues and cells has been studied mostly by biochemical and immunochemical analysis of lipid extracts separated by thin-layer chromatography. GSLs and gangliosides involved in the GM1b biosynthetic pathway were assigned to T-lymphocytes, whereas B-cell gangliosides and GSLs have been poorly characterized in former publications. We used specific polyclonal antibodies in immunohistochemistry and flow cytometry to analyze the distribution of globotriaosylceramide (Gb3Cer), globoside (Gb4Cer), gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), and gangliosides GM3 and GalNAc-GM1b in the mouse thymus, spleen, and lymph node. Immature thymocytes expressed epitopes recognized by all antibodies, except for anti-Gb4Cer. Mature thymocytes bound only antibodies to GalNAc-GM1b, Gg4Cer, and Gb4Cer. In secondary lymphoid organs, antibodies to globo-series GSLs bound to vascular spaces of secondary lymphoid organs, whereas the ganglio-series GSL antibodies recognized lymphocyte-containing regions. In a Western blotting analysis, only GalNAc-GM1b antibody recognized a specific protein band in all three organs. Flow cytometric analysis of spleen and lymph node cells revealed that B-cells carried epitopes recognized by all antibodies, whereas the T-cell GSL repertoire was mostly oriented to ganglio-series-neutral GSLs and GM1b-type gangliosides. The results of immunohistochemistry and flow cytometry were not always identical, possibly because of crossreactivity to glycoprotein-linked oligosaccharides and/or differences between cell surface carbohydrate profiles of isolated cells and cells in a tissue environment. (J Histochem Cytochem 48:1677–1689, 2000)
Key Words: flow cytometry, gangliosides, glycosphingolipids, immunohistology, in vivo, lymph node, mouse, spleen, thymus
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