地高辛标记的原位杂交法检测神经肽Y表达的条件优化
地高辛配基,,原位杂交;地高辛配基;神经肽Y;脑切片,0引言,1材料和方法,2结果和讨论,【参考文献】
Optimization of digoxigenin in situ hybridization detection of neuropeptide Y expressionWU YongFei, ZHANG JianHua, LI ShengBin
1State Key Laboratory of Forensic Sciences, Xian Jiaotong University College of Medicine, Xian 710061, China, 2Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267,USA
【Abstract】 AIM: To optimize Digoxigenin (Dig) in situ hybridization (ISH) detection of neuropeptide Y (NPY) expression in mouse hippocampus. METHODS: The Diglabeled RNA antisense and sense probes for NPY were prepared by in vitro transcription. Kainic acid (KA) was taken to induce NPY expression in the mouse hippocampus. Mice were sacrificed 2 hs after KA injection and sections of 12 μm thickness were used for analysis. The influence of different pH of the fixative, different fixation time, different prehybridization treatment, and different prehybridization and hybridization solutions were compared. RESULTS: Different pH of the fixative from 7.5 to 9.5 did not have noticeable influence on the hybridization signals. Thirtyminute fixation time was enough to obtain good result. Prehybridization treatment with Triton X100 or mild digestion by proteinase K (PK) resulted in reduced tissue preservation and signal intensity compared to the untreated section. Salmon sperm DNA of 40 mg/L or 250 mg/L of bakers yeast RNA was sufficient to avoid background problem as did Denhardts solution. CONCLUSION: A simple and effective protocol is developed for Dig ISH detection of NPY expression. ......
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