Perrio Cécile, Nicole Olivier, Buisson Alain
Normandie University, UNICAEN, CEA, CNRS, UMR6301-ISTCT, LDM-TEP, Cyceron, Caen, France.
Université de Bordeaux, CNRS UMR 5293, Institut des Maladies Neurodégénératives, Bordeaux, France.
Methods Mol Biol. 2017;1677:171-183. doi: 10.1007/978-1-4939-7321-7_9.
Laser Scanning Confocal Microscopy (LSCM) imaging using an appropriate fluorescent probe enables the visualization of a molecular target with high resolution, and represents a method of choice for studying expression, subcellular location, and trafficking of receptors in living cells. The chemical, physical, and pharmacological properties of the probe remain essential. Here, we describe (1) the preparation of a specific probe for NMDAR GluN2B receptor by conjugation of fluorescein to an ifenprodil-based ligand, (2) an in vitro functional assay by calcium imaging for GluN2B binding and inhibition evaluation of the probe, and (3) the labeling and confocal imaging of GluN2B in DS-red labeled living cortical neurons.
使用合适的荧光探针进行激光扫描共聚焦显微镜（LSCM）成像，能够以高分辨率可视化分子靶点，是研究活细胞中受体的表达、亚细胞定位和运输的首选方法。探针的化学、物理和药理性质仍然至关重要。在此，我们描述了：（1）通过将荧光素与基于艾芬地尔的配体偶联来制备用于NMDAR GluN2B受体的特异性探针；（2）通过钙成像进行体外功能测定，以评估该探针的GluN2B结合和抑制作用；以及（3）在DS-红色标记的活皮层神经元中对GluN2B进行标记和共聚焦成像。
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