Application of Automated mRNA In Situ Hybridization for Formalin-fixed, Paraffin-embedded Mouse Skin Sections: Effects of Heat and Enzyme Pretreatment on mRNA Signal Detection
Application of Automated mRNA In Situ Hybridization for Formalin-fixed, Paraffin-embedded Mouse Skin Sections: Effects of Heat and Enzyme Pretreatment on mRNA Signal Detection
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自动 mRNA 原位杂交在福尔马林固定、石蜡包埋的小鼠皮肤切片中的应用：热和酶预处理对 mRNA 信号检测的影响
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
T. Grogan
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文献类型：
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作者：
H. Nitta;J. Kishimoto;T. Grogan
最近，Ventana Discovery仪器引入了自动mRNA原位杂交应用。该应用程序的设计使得从烘烤到信号检测的所有必要步骤在仪器上在1天内完成。我们应用这种技术来可视化福尔马林固定的小鼠皮肤石蜡组织切片中多功能蛋白聚糖的表达位点。本研究的重点是证明蛋白酶消化或加热预处理，称为细胞调理，对杂交信号的影响，使用一个很好的特点versican反义核糖核酸探针。石蜡切片自动脱蜡、固定和酸处理。然后，对组织切片进行单独的蛋白酶消化（3种强度）、单独的细胞调节或细胞调节和蛋白酶消化的组合。用地高辛标记的多功能蛋白聚糖反义探针（20 ng/载玻片）杂交6小时，并在仪器上使用硝基蓝氯化四唑5-溴-4-氯-3-吲哚基磷酸甲苯胺盐（NBT/BCLIP）底物溶液检测信号3小时。单独的细胞调节不产生任何信号，而最高强度的蛋白酶消化产生明显的背景染色。然而，当细胞调理和温和的蛋白酶消化相结合，多功能蛋白聚糖mRNA的信号清楚地表明在毛乳头区域。因此，我们证明了与单独的蛋白酶消化或细胞预处理步骤相比，先进行细胞预处理步骤，然后进行温和的蛋白酶消化，可以增强mRNA靶染色。我们验证了自动化原位杂交过程适用于福尔马林固定的小鼠皮肤石蜡切片，并且自动化1天方案简单且可重复。自动化的精确控制允许微调温度和酶剂量，以找到信噪比和形态的最佳测定条件。
Recently, an automated mRNA in situ hybridization application was introduced for the Ventana Discovery instrument. The application was designed so that all necessary steps from baking through signal detection were completed within 1 day on the instrument. We applied this technology for visualizing the expression site of versican in formalin-fixed mouse skin paraffin tissue sections. Our focus of this study was to demonstrate the effects of protease digestion or heating pretreatment, termed cell conditioning, on the hybridization signal using a well characterized versican antisense riboprobe. Paraffin sections were automatically deparaffinized, fixed, and acid-treated. Then, the tissue sections were subjected to protease digestion alone (3 strengths), cell conditioning alone, or the combination of cell conditioning and protease digestion. Hybridization was performed with digoxigenin-labeled versican antisense probe (20 ng/slide) for 6 hours, and the signal was detected using a Nitro blue Tetrazolium chloride 5-Bromo-4-cloro-3-indolyl phosphate toluidine salt (NBT/BCLIP) substrate solution for 3 hours on the instrument. Cell conditioning alone did not produce any signal, whereas the highest strength of protease digestion produced noticeable background staining. However, when cell conditioning and mild protease digestion were combined, the signal for versican mRNA was clearly demonstrated in the hair papilla region. Thus, we demonstrated the effects of the cell conditioning step followed by mild protease digestion for enhancing the mRNA target staining compared with protease digestion or the cell conditioning step alone. We verified that the automated in situ hybridization process was applicable for formalin-fixed mouse skin paraffin sections and that the automated 1-day protocol is simple and reproducible. The precise control of automation allows fine tuning of temperature and enzyme dose to find the optimized assay condition for the signal to noise ratio and morphology.