Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy (Plant Methods)

This is a very interesting paper that provide a new, fast and easy protocol, with specific step-by-step instructions, for a trustworthy imaging with cellular resolution of fixed Arabidopsis ovules at different developmental stages. The authors combine two previously outlined techniques: clearing of fixed ovules in ClearSee solution, and marking the cell outline using the cell wall stain SCRI Renaissance 2200. A key point for the method is the staining with TO-PRO-3 that stains nuclei in fixed ovules, including the nuclei of the embryo sac. Moreover, TO-PRO-3 works well with SR2200, allowing the combination of cell wall and nuclear staining. The best results could be achieved with ClearSee, SR2200 and TO-PRO-3, followed by dissection and mounting the samples in Vectashield. Using the new protocol is possible to generate digital 3D models of ovules of various stages, providing the foundation for a future quantitative analysis of ovule morphogenesis. (Summarized by Francesca Resentini) Plant Methods 10.1186/s13007-019-0505-x