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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Identification of κ opioid receptors in the immune system by indirect immunofluorescence
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Identification of κ opioid receptors in the immune system by indirect immunofluorescence

机译：通过间接免疫荧光鉴定免疫系统中的κ阿片受体

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摘要

A method to visualize the κ opioid receptor is described that uses a high-affinity fluorescein-conjugated opioid ligand and indirect immunofluorescence with the phy-coerythrin fluorophore to amplify the signal. The mouse thymoma cell line R1E/TL8x.1.G1.OUA~r.1 (R1EGO), which expresses the κ_1 but not μ or δ opioid receptors, was used as a positive control for fluorescence labeling. A fluorescein isothiocyanate-conjugated arylacetamide (FITC-AA) compound displaying high affinity for the κ opioid receptor was synthesized. R1EGO cells were incubated with FITC-AA, in the absence or presence of the κ-selective opioid antagonist nor-binaltorphimine (nor-BNI) as a competitor. By using fluorescence microscopy and flow cytometry, incubation of R1EGO cells with FITC-AA alone was not sufficient for the detection of specific staining of the κ opioid receptor. To amplify the FITC-AA fluorescence, the fluorescein served as a hapten for subsequent antibody detection. R1EGO cells were incubated with FITC-AA, followed by biotinylated rabbit anti-fluorescein IgG and extravidin-conjugated R-phyco-erythrin. By using this approach, R1EGO cells were stained with phycoerythrin-amplif ied FITC-AA, and the staining was displaced with nor-BNI. Flow cytometry showed that titra-tions of both FITC-AA and nor-BNI produced saturable concentration-dependent changes in the median phyco-erythrin fluorescence intensity, with optimal staining at 30 μM FITC-AA. Up to 80% of the fluorescence above background was inhibited by nor-BNI. Freshly isolated thymocytes from C57BL/6ByJ mice also showed nor-BNI-sensitive staining with the FITC-AA amplification. This sensitive method of indirect phycoerythrin immunofluorescence can be used to amplify any fluorescein-conjugated opioid ligand for the detection of opioid receptors.
机译：描述了一种可视化κ阿片受体的方法，该方法使用高亲和力的荧光素共轭阿片配体和间接免疫荧光与藻红蛋白荧光团来放大信号。表达κ_1但不表达μ或δ阿片受体的小鼠胸腺瘤细胞系R1E / TL8x.1.G1.OUA〜r.1（R1EGO）被用作荧光标记的阳性对照。合成了对κ阿片受体具有高亲和力的异硫氰酸荧光素共轭芳基乙酰胺（FITC-AA）化合物。 R1EGO细胞与FITC-AA一起孵育，不存在或存在κ选择性阿片拮抗剂Nor-binaltorphimine（nor-BNI）作为竞争剂。通过使用荧光显微镜和流式细胞术，仅用FITC-AA孵育R1EGO细胞不足以检测κ阿片受体的特异性染色。为了扩增FITC-AA荧光，荧光素用作随后抗体检测的半抗原。将R1EGO细胞与FITC-AA孵育，然后进行生物素化的兔抗荧光素IgG和缀合有抗生物素蛋白的R-藻红蛋白进行孵育。通过这种方法，R1EGO细胞用藻红蛋白扩增的FITC-AA染色，然后用nor-BNI取代染色。流式细胞仪显示，FITC-AA和nor-BNI的滴定均会产生中位数藻红蛋白荧光强度的饱和浓度依赖性变化，且在30μMFITC-AA处具有最佳染色效果。 nor-BNI抑制了超过背景的多达80％的荧光。来自C57BL / 6ByJ小鼠的新鲜分离的胸腺细胞还显示出FITC-AA扩增对nor-BNI敏感的染色。这种敏感的间接藻红蛋白免疫荧光方法可用于扩增任何荧光素偶联的阿片样物质配体，以检测阿片样物质受体。

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