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.

Keywords: immunofluorescence; lncRNA; mRNA; mouse embryo; single-molecule RNA FISH; whole-mount.

FIGURE 1

A workflow diagram for (A) mouse embryo collection and fixation and (B) the IF and smRNA FISH protocol.

FIGURE 2

(A) Image represents no primary antibody control. DAPI stain to the nucleus. Image is shown at a single z-plane, scale bars: 12 μm. Images are shown of a no smRNA FISH probe control. DAPI stain to the nucleus. Images are shown at a single z-plane and maximum intensity z-projection, scale bars: 12 μm. (B) A negative-control probe targeting the E. coli dapB gene. DAPI stain to the nucleus. All the images are shown at a single z-plane and maximum intensity z-projection, scale bars: 12 μm. (C) Images are shown of a positive-control probe targeting the mouse Polr2a gene. The maximum intensity Z-projection of mouse blastocysts images are shown here, with scale bars: 12 μm.

FIGURE 3

(A) Combining IF and smRNA FISH using a Platr4 lncRNA probe and anti-Cdx2 antibody (B) Combining IF and smRNA FISH using a Platr4 lncRNA probe and anti-Tead4 antibody. (C) Combining IF and smRNA FISH using a Malat1 lncRNA probe and anti-Cdx2 antibody. (D) Combining IF and smRNA FISH using a Malat1 lncRNA probe and anti-Tead4 antibody. Nuclei were counterstained with DAPI. Mouse anti-Cdx2 and mouse anti-Tead4 primary and Alexa Fluor 488-conjugated goat anti-mouse secondary antibodies were used for IF detection. All the images are shown at a single z-plane, scale bars, 12 μm.

FIGURE 4

(A) Detection of multiple RNA transcripts, such as lncRNA Platr4, mRNA Pou5f1, and Cdx2 protein in 3D-mouse blastocysts. Nuclei were counterstained with DAPI. The green color represents the Cdx2 protein signal, and red and cyan punctate dots represent the RNA signal for Platr4 and Pou5f1 transcripts. All images are shown at the maximum intensity, the Z-projection, scale bars: 12 μm. (B) Detection of Malat1 lncRNA, Pou5f1 mRNA, and Cdx2 protein in 3D-mouse blastocysts. Nuclei were counterstained with DAPI. The green color represents the Cdx2 protein signal, and red and cyan punctate dots represent the RNA signal for Malat1 and Pou5f1 transcripts. All images are shown at the maximum intensity, the Z-projection, scale bars: 12 μm.