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Epifluorescence or confocal microscope with image capture software

Company: Leica
Catalog#: Leica AF6000 epifluorescence microscope
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Ligand and Carbohydrate Engagement (LACE) Assay and Fluorescence Quantification on Murine Neural Tissue
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2021-03-20
[Abstract]  

The interaction between cell surface heparan sulphate and diffusible ligands such as FGFs is of vital importance for downstream signaling, however, there are few techniques that can be used to investigate this binding event. The ligand and carbohydrate engagement (LACE) assay is a powerful tool which can be used to probe the molecular interaction between heparan sulphate and diffusible ligands and can detect changes in binding that may occur following genetic or pharmacological intervention. In this protocol we describe an FGF17:FGFR1 LACE assay performed on embryonic mouse brain tissue. We also describe the method we have used to quantify changes in fluorescent LACE signal in response to altered HS sulphation.

[摘要]  [摘要]细胞表面硫酸乙酰肝素与可扩散配体(例如FGFs)之间的相互作用对于下游信号传导至关重要,但是,很少有技术可用于研究这种结合事件。配体和碳水化合物结合(LACE)分析是一种功能强大的工具,可用于探测硫酸乙酰肝素与可扩散配体之间的分子相互作用,并可检测在遗传或药理学干预后可能发生的结合变化。在此协议中,我们描述了在胚胎小鼠脑组织上进行的FGF17:FGFR1 LACE分析。我们还描述了我们用来量化荧光LACE信号响应HS硫酸盐改变的变化的方法。


[背景]硫酸乙酰肝素(HS)是一种细胞外基质和细胞表面糖胺聚糖分子,可通过硫酸化进行广泛修饰。HS与包括FGF,Wnt,BMP和Slits在内的多种具有重要发展意义的信号分子相互作用。例如,在FGF信号传导期间,HS充当共受体,促进FGF配体与细胞表面FGFR受体的结合。此形成HS:FGF:FGFR需要复杂的对FGF信号发生(阿伦等人,2001) 。已显示HS的差异硫酸化会影响FGF配体与其FGFR细胞表面受体的结合。在我们最近的论文中,我们使用了配体和碳水化合物结合(LACE)分析方法来检测HS和FGF蛋白之间的相互作用(Clegg et ...

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