Jiang Hao, Feng Lei, Soriano del Amo David, Seidel Iii Ronald D, Marlow Florence, Wu Peng
Department of Biochemistry, Albert Einstein College of Medicine, Yeshiva University, USA.
J Vis Exp. 2011 Jun 6(52):2686. doi: 10.3791/2686.
Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid and N-acetylgalactosamine, in developing zebrafish and in other living organisms.
最近，通过用叠氮化物或炔烃标记的单糖处理细胞或生物体，利用生物正交化学报告策略实现了体内聚糖成像。经聚糖生物合成机制加工的修饰单糖被整合到细胞表面糖缀合物中。然后，生物正交叠氮化物或炔烃标签允许与荧光探针进行共价缀合以进行可视化，或与亲和探针进行共价缀合以进行富集和糖蛋白质组分析。本方案描述了通常用于斑马鱼胚胎中岩藻糖基化聚糖无创成像的程序，包括：1）将GDP-5-炔基岩藻糖（GDP-FucAl）显微注射到单细胞期胚胎中，2）通过生物相容性铜（I）催化的叠氮化物-炔烃环加成反应（CuAAC），用叠氮化物缀合的荧光团标记斑马鱼胚胎包被层中的岩藻糖基化聚糖，3）通过共聚焦显微镜成像。这里描述的方法可以很容易地扩展到在发育中的斑马鱼和其他活生物体中可视化其他类型的聚糖，例如含有唾液酸和N-乙酰半乳糖胺的聚糖。
