Whole-Mount In Situ Hybridization Using Microwave Treatment

In situ mRNA hybridization, which relies on the complementarity between antisense riboprobes and specific RNA populations, was originally used on thin sections with colorimetric dyes to visualize mRNA distribution. The use of fluorescent probes and confocal imaging enabled whole-mount in situ hybridization (WISH) and 3D reconstruction of gene expression domains. Here, we present a versatile protocol based on microwave treatment that results in high and homogeneous hybridization yields. When used with a cell wall marker and tissue clearing, it enables simultaneous visualization of gene expression and cell segmentation across various Arabidopsis organs, including leaves, shoots, roots, and embryos enclosed within integuments. This easy, rapid, robust, and low-cost method also accommodates different detection kits and cytological dyes, and it can be extended to Double WISH for the simultaneous detection of two distinct mRNA targets.