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.
Keywords: Alkaline phosphatase; Fast Blue BB; Fast Red; In Situ hybridization; RNA; calcein; immunofluorescence; xylenol orange.