Histological Staining and Hydrogen Peroxide Visualization of the Abscission Zone in Setaria viridis

The abscission zone (AZ) consists of specialized cell layers where cell separation or breakage occurs that result in organ detachment. Microscopic observation of the AZ is crucial for understanding its function. The AZ undergoes cellular and physiological changes prior to abscission, such as cell death, loss of chlorophyll, and the production of reactive oxygen species (ROS). These changes can be visualized using specific dyes and indicators under light or fluorescent microscopes. However, one challenge of using these dyes is their inefficient penetration into the tissue, especially when the epidermal layer has thick secondary cell walls. In this chapter, a detailed protocol to overcome this challenge is described. Using the fruit AZ of Setaria viridis, in which the epidermal cell wall is thick and lignified, we gently fix the dissected tissue, embed it in the Cryomatrix, and trim off the outer cell layers using a cryostat. The tissue with exposed inner cells can then be stained with fluorescent dyes to visualize organelles of interest, or 3,3'-diaminobenzidine (DAB) to visualize hydrogen peroxide accumulated in the tissue.