TECHNICAL NOTE |
Correspondence to: Anthony H. Futerman, Dept. of Membrane Research and Biophysics, Weizmann Inst. of Science, Rehovot 76100, Israel.
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Summary |
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Many studies have examined the localization of gangliosides using anti-ganglioside antibodies, although widely differing conclusions have been reached. We now demonstrate that the apparent localization of gangliosides can be greatly influenced by the fixation method. Using monoclonal antibody (MAb) A2B5 (which reacts with a variety of gangliosides), hippocampal neurons were labeled at the cell surface when incubated with the antibody before fixation, but when incubated after fixation the cells displayed a variety of labeling patterns, depending on the fixation method. Biochemical analysis demonstrated that some of the fixatives (particularly acetone and methanol) significantly reduced or completely depleted cellular gangliosides, implying that the immunoreactivity observed with A2B5, and with other antibodies, was not due to gangliosides. When neurons were incubated with an anti-GD1b antibody prefixation, uniform labeling of the plasma membrane was observed, but after ganglioside depletion using biochemical inhibitors of ganglioside synthesis no cell surface labeling was detected. However, even in cells depleted of gangliosides, labeling of both the cell surface and intracellular compartments was observed when the anti-GD1b antibody was applied after fixation. Moreover, after fixation, antibodies to GM4 and GD2 reacted with hippocampal neurons, although these gangliosides are absent from these neurons. In contrast, the JONES antibody (which reacts with 9-O-acetylated GD3) labeled neurons with a similar pattern, essentially irrespective of the fixation method. These observations demonstrate that great care must be taken in assigning gangliosides to specific cell populations or to intracellular locations solely on the basis of use of anti-ganglioside antibodies, and suggest that optimal fixation conditions must be established for each anti-ganglioside antibody. (J Histochem Cytochem 45:611-618, 1997)
Key Words: gangliosides, immunofluorescence, neurons, fixatives, anti-ganglioside antibodies, fumonisin B1
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Introduction |
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The sialic acid-containing glycosphingolipids, the gangliosides, may play important roles in both neuronal (
Many studies have attempted to determine the cellular localization of gangliosides using anti-ganglioside antibodies (reviewed in
We now report that the immunolocalization of gangliosides can be greatly influenced by the fixation techniques used, at least in cultured hippocampal neurons, which contain high levels of gangliosides expressed at the cell surface (
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Materials and Methods |
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Cell Culture and Reagents
Hippocampal neurons (taken from embryonic Day 18 rats) were cultured as described for morphological (
Antibodies
The following MAbs were used in this study: A2B5 (Boeh-ringer-Mannheim; Mannheim, Germany), which reacts with a variety of polysialo-gangliosides (see
Indirect Immunofluorescence Microscopy
Coverslips containing neurons from 0-, 3-, and 7-day-old cultures were washed in Hank's balanced salt solution (HBSS) containing 3% (w/v) bovine serum albumin for 5 min: Then the neurons were either incubated directly with primary antibodies (37C, 30 min) and then fixed with 4% formaldehyde (either freshly depolymerized from paraformaldehyde, or frozen aliquots) for 30 min at 37C, or fixed before incubation (37C, 30 min) with the primary antibodies. The following fixation methods were used: (a) 4% formaldehyde, 30 min, 37C; (b) methanol, 10 min, -20C; (c) acetone, 1 min, -20C; (d) 4% formaldehyde, 20 min, 37C, followed by acetone, 1 min, -20C. MAbs B17, JONES, and R24, and the polyclonal anti-GM4 antibody were diluted 1:50, BMA190 was diluted 1:1, and A2B5 was diluted 1:100, all in HBSS containing 3% bovine serum albumin. Biotin-conjugated anti-rabbit or anti-mouse IgG (H+L) antibodies and indocarbocyanine (Cy3)-conjugated streptavidin were used for detection. Cells were observed with a Plan Apochromat x63/1.4 NA oil objective of a Zeiss Axiovert 35 microscope with an appropriate filter for Cy3 fluorescence.
Biochemical Analysis of Ganglioside Synthesis and Content
Gangliosides were metabolically labeled by incubation with 5 x 106 cpm of [4,5-3H]-dihydrosphinganine (10 Ci/mmol) (
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Results |
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MAb A2B5 reacts with a number of gangliosides, including GT3, 9-O-acetylated-GT3, GQ1b, GD3, GD2, GQ1c, sulfatide, and several minor gangliosides (
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To analyze whether the labeling observed after each fixation method was due to endogenous gangliosides, neurons were metabolically labeled with [4,5-3H]-dihydrosphinganine to label sphingolipids and gangliosides, and then fixed by the four methods used above (Figure 1). To ensure no loss of cellular sphingolipids and gangliosides, neurons were scraped directly into distilled water and then lyophilized (
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We next compared the binding of a highly specific MAb, B17 (which binds to ganglioside GD1b), to hippocampal neurons after various fixation methods. When 3-day-old neurons were incubated with B17 before fixation, uniform labeling of the cell surface was observed (Figure 3A and Figure 3B). However, after incubation with either PDMP (Figure 3C and Figure 3D) or FB1 (Figure 4A and Figure 4B), both of which inhibit ganglioside synthesis (
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Whereas no labeling with B17 was seen when neurons were incubated with the antibody before formaldehyde fixation in PDMP- or FB1-treated cells (Figure 3C, Figure 3D, Figure 4A, and 4B), a variety of labeling patterns were seen after incubation with the antibody after fixation, including striped labeling in the neuronal processes (formaldehyde fixation; Figure 4C and Figure 4D), bright labeling of the whole cell (formaldehyde and acetone fixation; Figure 4E and Figure 4F), labeling of the cell body and nuclear envelope (acetone fixation; Figure 4G and Figure 4H), and labeling of the cell body (methanol; Figure 4I and Figure 4J). Because GD1b levels were almost completely abolished by PDMP and FB1 treatment, these labeling patterns cannot reflect the actual intra-cellular location of GD1b. Likewise, a striped labeling pattern was also observed when formaldehyde-fixed neurons were incubated with a polyclonal antibody to GM4 (Figure 5B) and with MAb BMA190, which reacts with GD2 (Figure 5D), although when incubated with the antibody before formaldehyde fixation no labeling was observed (Figure 5A and Figure 5C). Because GD2 is expressed mainly in undifferentiated tissues and in tumor cells (
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In contrast to the variety of labeling patterns observed with different fixation methods for the four antibodies discussed above, the labeling patterns observed with the JONES antibody (which reacts with 9-O-acetylated GD3) (Figure 6), or with antibody R24 (which reacts with ganglioside GD3) (not shown), were not affected by fixation. JONES labeled 30% of neurons 2 hr after neurons were plated on glass coverslips (Figure 7A), and a similar number of JONES-positive cells and a similar pattern of labeling were observed after all fixation techniques (Figure 6C-H), with the exception of methanol, which completely abolished labeling (Figure 6I and Figure 6J). The number of JONES-positive cells decreased significantly as neurons matured, and labeling was most prominent in cells that had not yet extended neuronal processes (Figure 7A). This is consitent with previous studies that used this antibody to examine the developmental profile and cellular localization of 9-O-acetylated GD3 (
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Discussion |
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Anti-ganglioside antibodies have become a popular tool for localizing gangliosides in neuronal and non-neuronal tissues (
Organic solvents, such as acetone and methanol, are particularly problematic as fixatives because both solubilize gangliosides to various extents. Our biochemical analysis demonstrated that methanol completely solubilizes gangliosides from hippocampal neurons, with acetone also solubilizing gangliosides by >50%. This implies that binding of anti-ganglioside antibodies to methanol-treated tissues may be due to crossreactivity of the antibodies with unrelated epitopes, and also suggests that published conclusions using acetone as a fixative may need to be re-evaluated. It has been suggested that gangliosides become antigenic only after acetone treatment, because acetone may act to promote the accessibility of the ganglioside (Graus et al. 1994) or may unmask membrane-bound carbohydrates (
Mild fixatives such as formaldehyde can also cause artifactual labeling. Therefore, an anti-GM4 antibody bound to formaldehyde-fixed hippocampal neurons, even though GM4 is absent from these neurons and is found only in glia (
The reason for the differences in the sensitivity of various antibodies to different fixatives is not clear, but may be due to differences in the solubility of the glycolipid antigens, to differences in accessibility, or to nonspecific binding of certain antibodies to related carbohydrate antigens that may become accessible after treatment with various fixatives. However, it is clear that the differences are not due to the immunoglobulin class, because both B17 and JONES antibodies are of the IgM class but each antibody behaves differently under various fixation conditions.
In summary, whereas previous workers have recognized that the antigenicity and immunogenicity of gangliosides and other glycosphingolipids depends on the density of the antigen on the cell surface, the chain length of the fatty acids and their -hydroxylation, and the cryptic behavior of glycosphingolipids (
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Acknowledgments |
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Supported by the German-Israel Foundation for Scientific Research and Development.
We thank Dr Israel Pecht for providing the B17 antibody, Dr Colin J. Barnstable for providing the JONES antibody, and Behring Werke AG for providing BMA190. We also thank Rivi Zisling for expert help in preparing and maintaining hippocampal cultures. AHF is the incumbent of the Recanati Career Development Chair in Cancer Research.
Received for publication September 26, 1996; accepted November 22, 1996.
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Literature Cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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Bernhard H, Roth S, Bauerschmitz J, Meyer zum Büschenfelde K-H, Dippold W (1992) Immunorecognition of different ganglioside epitopes on human normal and melanoma tissues. Int J Cancer 51:568-572[Medline]
Blum AS, Barnstable CJ (1987) O-acetylation of cell-surface carbohydrate creates discrete molecular patterns during neuronal development. Proc Natl Acad Sci USA 84:8716-8720[Abstract]
Constantine-Paton M, Blum AS, Mendez-Otero R, Barnstable CJ (1986) A cell surface molecule distributed in a dorsoventral gradient in the perinatal rat retina. Nature 324:459-462[Medline]
Daniotti JL, Landa CA, Maccioni HJ (1994) Regulation of ganglioside composition and synthesis is different in developing chick retinal pigment epithelium and neural retina. J Neurochem 62:1131-1136[Medline]
Dippold WG, Lloyd KO, Li LTC, Ikeda H, Oettgen HF, Old LJ (1980) Cell surface antigens of human malignant melanoma: definition of six antigenic systems with mouse monoclonal antibodies. Proc Natl Acad Sci USA 77:6114-6118[Abstract]
Dotti CG, Sullivan CA, Banker GA (1988) The establishment of polarity by hippocampal neurons in culture. J Neurosci 8:1454-1468[Abstract]
Fredman P, Magnani JL, Nirenberg M, Ginsburg V (1984) Monoclonal antibody A2B5 reacts with many gangliosides in neuronal tissue. Arch Biochem Biophys 233:661-666[Medline]
Futerman AH (1994) An update on sphingolipid synthesis and transport along the secretory pathway. Trends Glycosci Glycotech 6:143-153
Goldman JE, Hirano M, Yu RK, Seyfried TN (1984) GD3 ganglioside is a glycolipid characteristic of immature neuroectodermal cells. J Neuroimmunol 7:179-192[Medline]
Goslin K, Banker G (1991) Rat hippocampal neurons in low-density culture. In Banker G, Goslin K, eds. Culturing Nerve Cells. Cambridge, MA, MIT Press, 251-281
Graus F, Cordon CC, Houghton AN, Melamed MR, Old LJ (1984) Distribution of the ganglioside GD3 in the human nervous system detected by R24 mouse monoclonal antibody. Brain Res 324:190-194[Medline]
Hakomori S (1986) Tumor-associated glycolipid antigens, their metabolism and organisation. Chem Phys Lipids 42:209-233[Medline]
Hakomori S, Igarashi Y (1993) Gangliosides and glycosphingolipids as modulators of cell growth, adhesion, and transmembrane signaling. Adv Lipid Res 25:147-162[Medline]
Harel R, Futerman AH (1993) Inhibition of sphingolipid synthesis affects axonal outgrowth in cultured hippocampal neurons. J Biol Chem 268:14476-14481
Hirschberg K, Rodger J, Futerman AH (1993) The long-chain sphingoid base of sphingolipids is acylated at the cytosolic surface of the endoplasmic reticulum in rat liver. Biochem J 290:751-757[Medline]
Hirschberg K, Zisling R, van Echten-Deckert G, Futerman AH (1996) Ganglioside synthesis during the development of neuronal polarity: major changes occur during axonogenesis and axon elongation, but not during dendrite growth or during synapto-genesis. J Biol Chem 271:14876-14882
Ledeen RW, Wu G (1992) Ganglioside function in the neuron. Trends Glycosci Glycotech 4:174-187
Lloyd KO, Gordon CM, Thampoe IJ, DiBenedetto C (1992) Cell surface accessibility of individual gangliosides in malignant melanoma cells to antibodies is influenced by the total ganglioside composition of the cells. Cancer Res 52:4948-4953[Abstract]
Matyas GR, Morre DJ (1987) Subcellular distribution and biosynthesis of rat liver gangliosides. Biochim Biophys Acta 921:599-614[Medline]
Merrill AH, Liotta DC, Riley RT (1996) Fumonisins: fungal toxins that shed light on sphingolipid function. Trends Cell Biol 6:218-223
Ortega E, Licht A, Biener Y, Pecht I (1990) A glycolipid-specific monoclonal antibody modulates Fce receptor stimulation by mast cells. Mol Immunol 27:1269-1277[Medline]
Prasadarao N, Tobet SA, Jungalwala FB (1990) Effect of different fixatives on immunocytochemical localization of HNK-1-reactive antigens in cerebellum: a method for differentiating the localization of the same carbohydrate epitope on proteins vs lipids. J Histochem Cytochem 38:1193-1200[Abstract]
Pukel CS, Lloyd KO, Travassos LR, Dippold WR, Oettgen HF, Old LJ (1982) GD3a prominent ganglioside of human melanoma: detection and characterization by mouse monoclonal antibody. J Exp Med 155:1133-1147[Abstract]
Radin NS, Vunnam RR (1981) Inhibitors of cerebroside metabolism. Methods Enzymol 72:673-684[Medline]
Reynolds R, Wilkin GP (1988) Expression of GD3 ganglioside by developing rat cerebellar Purkinje cells in situ. J Neurosci Res 20:311-319[Medline]
Schwarz A, Futerman AH (1996) The localization of gangliosides in the brain using anti-ganglioside antibodies. Biochim Biophys Acta 1286:247-267[Medline]
Schwarz A, Rapaport E, Hirschberg K, Futerman AH (1995) A regulatory role for sphingolipids in neuronal growth: inhibition of sphingolipid synthesis and degradation have opposite effects on axonal branching. J Biol Chem 270:10990-10998
Sofer A, Futerman AH (1995) Cationic amphiphilic drugs inhibit the internalization of cholera toxin to the Golgi apparatus and the subsequent elevation of cyclic AMP. J Biol Chem 270:12117-12122
Stein O, Stein Y (1971) Light and electron microscopic radioautography of lipids: techniques and biological applications. Adv Lipid Res 9:1-72[Medline]
Thorpe SJ, Kerr MA (1994) Common immunological techniques: ELISA, blotting, immunohistochemistry and immunocytochemistry. In Thorpe SJ, Kerr MA, eds. Immunochemistry. Oxford, Bios Scientific, 175-209
van Echten G, Sandhoff K (1993) Ganglioside metabolism. Enzymology, topology, and regulation. J Biol Chem 268:5341-5344
Williams MA, McCluer RH (1980) The use of Sep-PakTM C18 cartridges during the isolation of gangliosides. J Neurochem 35:266-269[Medline]
Yu RK, Iqbal K (1979) Sialosylgalactosyl ceramide as a specific marker for human myelin and oligodendroglia perikarya gangliosides of human myelin, oligodendroglia and neurons. J Neurochem 32:293-300[Medline]
Yu RK, Macala LJ, Farooq M, Sbaschnig AM, Norton WT, Ledeen RW (1989) Ganglioside and lipid composition of bulk-isolated rat and bovine oligodendroglia. J Neurosci Res 23:136-141[Medline]
Yu RK, Saito M (1989) Structure and localization of gangliosides. In Margolis RU, Margolis RK, eds. Neurobiology of Glycoconjugates. New York, Plenum Press, 1-42
Zeller CB, Marchase RB (1992) Gangliosides as modulators of cell function. Am J Physiol 262:C1341-C1355