Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy

Abstract

Whole-mount single-molecule RNA fluorescence in situ hybridization (smRNA FISH) in combination with immunofluorescence (IF) offers great potential to study long non-coding RNAs (lncRNAs): their subcellular localization, their interactions with proteins, and their function. Here, we describe a step-by-step, optimized, and robust protocol that allows detection of multiple RNA transcripts and protein molecules in whole-mount preimplantation mouse embryos. Moreover, to simultaneously detect protein and enable RNA probe penetration for the combined IF/smRNA FISH technique, we performed IF before smRNA FISH. We removed the zona pellucida, used Triton X-100 to permeabilize the embryos, and did not use a proteinase digestion step so as to preserve the antigens. In addition, we modified the IF technique by using RNase-free reagents to prevent RNA degradation during the IF procedure. Using this modified sequential IF/smRNA FISH technique, we have simultaneously detected protein, lncRNA, and mRNA in whole-mount preimplantation embryos. This reliable and robust protocol will contribute to the developmental biology and RNA biology fields by providing information regarding 3D expression patterns of RNA transcripts and proteins, shedding light on their biological function.

High-sensitivity whole-mount in situ hybridization of mouse oocytes and embryos visualizes the super-resolution structures and distributions of mRNA molecules

“…We developed a whole-mount in situ hybridization method with high sensitivity and super-resolution. This method enables analysis of the mRNA accumulation in a simple, convenient and cost-effective manner, compared with section in situ hybridization combined with the TSA system [23] and whole-mount smRNA-FISH with the RNA-scope technique [27][28][29].…”

“…Recent studies have shown that a single molecule RNA uorescent in situ hybridization (smRNA-FISH) with the RNA-scope technique enables detection of mRNAs in mammalian oocytes and embryos with high sensitivity in whole-mount samples [27][28][29]. In these methods, mRNAs are detected by the hybridization of Z-shaped DNA probes consisting of 50-60 bases, of which 20-25 bases on one side are complementary to sequences of the speci c transcripts and 14 bases on the other side are complementary to pre-ampli er DNA sequences.…”

Identification of glioblastoma stem cell-associated lncRNAs using single-cell RNA-sequencing datasets

“…Single-molecule RNA FISH was performed according to the manufacturer’s protocol for the RNAscope® Fluorescent Multiplex Reagent Kit 320850 (ACD #320850), as described previously [45, 46]. Briefly, 5 × 10 4 GSCs were seeded onto laminin-coated coverslips (12 mm) in a 24-well plate for 24 hours to reach 80% confluence, then fixed in freshly prepared 4% paraformaldehyde (PFA) (Electron Microscopy Sciences, 19200).…”