A ClearSee-Based Clearing Protocol for 3D Visualization of Arabidopsis thaliana Embryos

Abstract

Tissue clearing methods combined with confocal microscopy have been widely used for studying developmental biology. In plants, ClearSee is a reliable clearing method that is applicable to a wide range of tissues and is suitable for gene expression analysis using fluorescent reporters, but its application to the Arabidopsis thaliana embryo, a model system to study morphogenesis and pattern formation, has not been described in the original literature. Here, we describe a ClearSee-based clearing protocol which is suitable for obtaining 3D images of Arabidopsis thaliana embryos. The method consists of embryo dissection, fixation, washing, clearing, and cell wall staining and enables high-quality 3D imaging of embryo morphology and expression of fluorescent reporters with the cellular resolution. Our protocol provides a reliable method that is applicable to the analysis of morphogenesis and gene expression patterns in Arabidopsis thaliana embryos.

Code and data availability

The paper's supplementary materials include Movie S1, the Z-stack confocal image data (157 serial optical sections) used for the paper's 3D embryo visualization analysis, publicly available at the MDPI supplementary URL. No author analysis code or trained models are reported.