Heffer-Lauc Marija, Viljetić Barbara, Vajn Katarina, Schnaar Ronald L, Lauc Gordan
Department of Medical Biology, University of Osijek School of Medicine, J. Huttlera 4, 31000, Osijek, Croatia.
J Histochem Cytochem. 2007 Aug;55(8):805-12. doi: 10.1369/jhc.7A7195.2007. Epub 2007 Apr 4.
Gangliosides and glycosylphosphatidylinositol (GPI)-anchored proteins contain lipid tails that tether them to the outer side of the cell membrane. This mode of association with the cell membrane enables them to take part in the organization of lipid rafts, but it also permits gangliosides and GPI-anchored proteins to be actively released from one cell and inserted into the membrane of another cell. Recently, we reported that under conditions of lipid raft isolation, Triton X-100 causes significant redistribution of both gangliosides and GPI-anchored proteins. Aiming to find a less disruptive detergent, we evaluated the effects of CHAPS, Saponin, deoxycholic acid, Trappsol, Tween 20, Triton X-100, Brij 96V, Brij 98, and SDS on brain tissue sections. At room temperature, all detergents (1% concentration) extracted significant amounts of both gangliosides and Thy-1. At 4C, the extraction was weaker, but Triton X-100, CHAPS, and deoxycholic acid caused significant redistribution of GD1a and Thy-1 from gray matter into the white matter. Both redistribution and extraction were significantly augmented when sections were incubated with detergents in the presence of primary antibodies. Of the nine tested detergents, none is the ideal choice. However, Brij 96V appears to be able to sufficiently reveal myelin epitopes while causing the least amount of artifacts. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.
神经节苷脂和糖基磷脂酰肌醇（GPI）锚定蛋白含有脂质尾巴，可将它们拴在细胞膜外侧。这种与细胞膜的结合方式使它们能够参与脂筏的组织，但也使神经节苷脂和GPI锚定蛋白能够从一个细胞中被主动释放并插入到另一个细胞的膜中。最近，我们报道在脂筏分离的条件下，Triton X-100会导致神经节苷脂和GPI锚定蛋白都发生显著的重新分布。为了找到一种干扰性较小的去污剂，我们评估了CHAPS、皂角苷、脱氧胆酸、Trappsol、吐温20、Triton X-100、Brij 96V、Brij 98和SDS对脑组织切片的影响。在室温下，所有去污剂（浓度为1%）都提取了大量的神经节苷脂和Thy-1。在4℃时，提取作用较弱，但Triton X-100、CHAPS和脱氧胆酸导致GD1a和Thy-1从灰质显著重新分布到白质中。当切片在一抗存在下与去污剂孵育时，重新分布和提取都显著增强。在这九种测试的去污剂中，没有一种是理想的选择。然而，Brij 96V似乎能够在产生最少伪影的同时充分显示髓磷脂表位。本文稿包含在线补充材料，网址为http://www.jhc.org。请在线访问本文以查看这些材料。
