Alkaline phosphatase-based chromogenic and fluorescence detection method for BaseScope™ In Situ hybridization

Wang, Yongfu; Xu, Wenjing; Maddera, Lucinda; Tsuchiya, Dai; Thomas, Nancy; Yu, C. Ron and Parmely, Tari (2019). Alkaline phosphatase-based chromogenic and fluorescence detection method for BaseScope™ In Situ hybridization. Journal of Histotechnology, 42(4) pp. 193–201.

DOI: https://doi.org/10.1080/01478885.2019.1620906

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.

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