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

Hazra, Rasmani, Spector, David L (October 2022) Simultaneous visualization of RNA transcripts and proteins in whole-mount mouse preimplantation embryos using single-molecule fluorescence in situ hybridization and immunofluorescence microscopy. Frontiers in Cell and Developmental Biology, 10. ISSN 2296-634X

PDF 2022-Spector-Simultaneous-visualization-of-RNA-transcripts-and-proteins.pdf Available under License Creative Commons Attribution. Download (2MB)

Abstract

<jats:p>Whole-mount single-molecule RNA fluorescence <jats:italic>in situ</jats:italic> 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.</jats:p>

Item Type:	Paper
CSHL Authors:	Spector, David L.
Communities:	CSHL labs > Spector lab CSHL Cancer Center Program CSHL Cancer Center Program > Gene Regulation and Inheritance Program CSHL Cancer Center Shared Resources > Animal Services CSHL Cancer Center Shared Resources > Microscopy Service
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	4 October 2022
Date Deposited:	27 Oct 2022 02:15
Last Modified:	09 Feb 2024 18:55
PMCID:	PMC9577017
URI:	https://repository.cshl.edu/id/eprint/40742

Actions (login required)

Administrator's edit/view item