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

SUBMITTER: Hazra R 

PROVIDER: S-EPMC9577017 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature