Staining and Clearing of Arabidopsis Reproductive Tissue for Imaging of Fluorescent Proteins

Methods Mol Biol. 2017;1669:87-94. doi: 10.1007/978-1-4939-7286-9_8.


Imaging of fluorescent proteins in whole-mount tissue is a powerful tool to understand growth and developmental processes, not only in plants. With the advent of genetically encoded fluorescent reporters, which specifically label reproductive cells in Arabidopsis, deep tissue imaging has become increasingly important for the study of plant reproduction. To penetrate the surrounding layers of maternal tissue, however, the tissue has to be cleared by homogenizing the refractive index of the sample, often leading to inactivation of fluorescent proteins. 2,2'-thiodiethanol (TDE) has recently been introduced as a clearing agent that allows the imaging of fluorescent proteins in a cleared plant tissue. Here, we describe a simple protocol that combines TDE-based tissue clearing with cell wall staining to outline cells that enable deep tissue imaging in reproductive structures of Arabidopsis thaliana.

Keywords: 2,2′-Thiodiethanol; 2-Photon microscopy; Arabidopsis thaliana; Confocal microscopy; Deep tissue imaging; Embryogenesis; Optical clearing; Pollen tube; Renaissance SR2200.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / metabolism*
  • Luminescent Proteins / metabolism*
  • Microscopy, Confocal / methods
  • Pollen Tube / metabolism
  • Reproduction / physiology*
  • Sulfhydryl Compounds / chemistry


  • Luminescent Proteins
  • Sulfhydryl Compounds
  • 2,2'-thiodiethanol