ABSTRACT The BaseScope™ assay is a novel, highly sensitive RNA in situ hybridization (ISH) technique, allowing detection of short RNA sequences as well as discrimination between single-nucleotide alterations. Multiplexing BaseScope™ ISH with immunofluorescence assay has proven challenging because the diffusion of colorimetric dyes such as Fast Red in aqueous solutions degrades spatial resolution. In this study, we explore alkaline phosphatase-based fluorescent signal detection methods and integrate it with BaseScope™ RNA ISH. We found that Fast Blue BB/NAMP can be used in BaseScope™ ISH for signal detection. Additionally, we found that the calcium binding fluorochromes calcein and xylenol orange can be used to localize alkaline phosphatase activity in both immunohistochemistry (IHC) and BaseScope™ ISH assays. When applied to mouse brain and intestine tissue sections, we successfully detected circular RNA molecules and cell proliferation antigen Ki-67 proteins. This technological advance expanded the substrate selection of alkaline phosphatase-based BaseScope™ RNA ISH to allow researchers and clinical professionals accurately assess RNA targets with immunofluorescent multiplexing.

中文翻译：

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基于碱性磷酸酶的显色和荧光检测方法，用于 basescope™ 原位杂交

摘要 BaseScope™ 检测是一种新型的、高度灵敏的 RNA 原位杂交 (ISH) 技术，可以检测短 RNA 序列以及区分单核苷酸变化。事实证明，将 BaseScope™ ISH 与免疫荧光分析相结合具有挑战性，因为比色染料（如固红）在水溶液中的扩散会降低空间分辨率。在本研究中，我们探索了基于碱性磷酸酶的荧光信号检测方法，并将其与 BaseScope™ RNA ISH 相结合。我们发现 Fast Blue BB/NAMP 可用于 BaseScope™ ISH 进行信号检测。此外，我们发现钙结合荧光染料钙黄绿素和二甲苯酚橙可用于在免疫组织化学 (IHC) 和 BaseScope™ ISH 检测中定位碱性磷酸酶活性。当应用于小鼠脑和肠组织切片时，我们成功检测到环状 RNA 分子和细胞增殖抗原 Ki-67 蛋白。这一技术进步扩大了基于碱性磷酸酶的 BaseScope™ RNA ISH 的底物选择，使研究人员和临床专业人员能够通过免疫荧光多重检测准确评估 RNA 靶标。