Fluorescence in situ hybridization (FISH) has offered an eloquent method for the detection of mRNA in whole-mount samples or sections for over 50 years. While low signal-to-background ratio was initially a limiting factor for FISH in certain fields, over the past two decades the incorporation of isothermal signal amplification mechanisms termed the hybridization chain reaction (HCR) has expanded FISH use into more diverse applications. After several iterations, the current third-generation in situ hybridization chain reaction (V3HCR) now allows clear detection of mRNA to subcellular or even single-molecule resolution. However, widespread lipofuscin autofluorescence, most often found in neuronal cell populations, interferes with HCR signal and can complicate analysis….
In a recent Current Protocols publication, May-Zhang et al. report a V3HCR protocol that allows the successful subcellular detection of specific mRNAs in mouse and human tissue cryosections. The protocol was optimized targeting mRNA in neuronal cells in adult intestinal tissue sections, but it can be applied to a range of tissues and sample thicknesses. After testing a panel of lipofuscin quenching methods, including quenching with cupric sulfate, bleaching in Dent’s fixative, detergent extraction, Murray’s clearing, treatment with 8% SDS, and standard Sudan Black blocking, Biotium’s TrueBlack® Lipofuscin Autofluorescence Quencher was determined to be the only acceptable option for overcoming lipofuscin autofluorescence without causing V3HCR signal loss.