Optimized Methods of Investigating Developmental Protein and Transcript Expression in Whole-mount Mouse Embryos

Mouse models have been instrumental in advancing our understanding of embryonic development. Analyzing the expression and localization of proteins and transcripts within embryos is critical for elucidating cellular and molecular mechanisms underlying development and disease. However, traditional techniques used to analyze transcripts and proteins in mouse embryos are limited. RNA in situ hybridization permits whole-mount staining of transcripts but lacks multiplexing capability. By contrast, sectioning of embryos allows for multiplexed target staining, but only in two-dimensions (2D). More recently, techniques such as RNAscope and immunofluorescence staining have been applied to whole-mount embryos, allowing multiplexing of targets in three-dimensions (3D). These methods enable mapping of key interactions, pathways, and cell types necessary for embryonic development without the limitations of traditional techniques. Here we describe optimized methods for whole-mount staining of fixed embryonic day (E) 8.5 and E9.5 mouse embryos using either RNAscope or immunofluorescence. Both enable multiplexed detection of up to 4 targets simultaneously in a single embryo. We also provide a method to combine immunofluorescence staining with RNAscope in the same embryo to visualize the interplay between proteins and transcripts during development. Furthermore, we describe a pipeline of confocal imaging and 3D analysis of target expression across the whole embryo and within defined developmental cell types using Zeiss Arivis Pro software. Together, these methods allow comprehensive analysis of multiplexed protein and transcript targets in 3D, providing a powerful system for dissecting cellular and molecular interactions in early to mid-embryogenesis.