Optimization of digoxigenin in situ hybridization detection of neuropeptide Y expression

    WU Yongª²Fei, ZHANG Jianª²Hua, LI Shengª²Bin

    1State Key Laboratory of Forensic Sciences, Xi¬ðan Jiaotong University College of Medicine, Xi¬ðan 710061, China, 2Department of Cell Biology, Neurobiology and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267,USA

    ¡¾Abstract¡¿ AIM: To optimize Digoxigenin (Dig) in situ hybridization (ISH) detection of neuropeptide Y (NPY) expression in mouse hippocampus. METHODS: The Digª²labeled RNA antiª²sense 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 ¦Ìm thickness were used for analysis. The influence of different pH of the fixative, different fixation time, different preª²hybridization treatment, and different preª²hybridization 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. Thirtyª²minute fixation time was enough to obtain good result. Preª²hybridization treatment with Triton Xª²100 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¬ðs solution. CONCLUSION: A simple and effective protocol is developed for Dig ISH detection of NPY expression. ......