Alkaline phosphatase-based chromogenic and fluorescence detection method for basescope (TM) In Situ hybridization

摘要

The BaseScope (TM) 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 (TM) 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 (TM) RNA ISH. We found that Fast Blue BB/NAMP can be used in BaseScope (TM) 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 (TM) 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 (TM) RNA ISH to allow researchers and clinical professionals accurately assess RNA targets with immunofluorescent multiplexing.