地高辛标记的原位杂交法检测神经肽Y表达的条件优化

Optimization of digoxigenin in situ hybridization detection of neuropeptide Y expression

目的 :优化用地高辛标记的原位杂交法检测小鼠海马结构中神经肽Y的表达 .方法 :用体外转录法获得地高辛标记的神经肽Y的反义和正义RNA探针 .用kainicacid诱导小鼠脑组织中神经肽Y表达 ,2h后牺牲小鼠 ,制备 1 2 μm厚的海马组织切片 .然后比较不同pH值 (7.5～ 9.5 )的固定剂 ,不同的固定时间 ,不同的杂交前预处理 ,以及不同预杂交及杂交液对杂交信号的影响 .结果 :从pH 7.5～ 9.5 ,不同碱性固定液对杂交信号没有明显影响 .30min的固定时间足以获得满意的杂交结果 .用蛋白酶K部分消化切片或用TritonX 1 0 0打孔反而降低了杂交信号 .4 0mg/LSalmonspermDNA或 2 5 0mg/LbakersyeastRNA可获得与Denhardt s杂交液相同的杂交信号 .结论 :通过优化 ,我们获得了一种简便而又有效的地高辛标记的原位杂交法 。

AIM: To optimize Digoxigenin (Dig) in situ hybridization (ISH) detection of neuropeptide Y (NPY) expression in mouse hippocampus. METHODS: The Dig labeled RNA anti sense 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 pre hybridization treatment, and different pre hybridization 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. Thirty minute fixation time was enough to obtain good result. Pre hybridization treatment with Triton X 100 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 Denhardts solution. CONCLUSION: A simple and effective protocol is developed for Dig ISH detection of NPY expression.