Protocol to fluorescently stain vacuoles in Arabidopsis root cells

Plant vacuoles are essential organelles that respond to developmental and environmental signals. Here, we present a protocol for staining the vacuole lumen in Arabidopsis root cells, enabling precise visualization of vacuolar dynamics. We describe steps for preparing plant material and staining with commonly used fluorescent dyes. We then detail procedures for visualizing vacuoles in the blue, green, and red emission spectra, allowing for their combined use with a variety of compatible fluorescent-tagged protein markers. For complete details on the use and execution of this protocol, please refer to Dubrovsky et al.,¹ Fricker,² Bassil et al.,³ Grzam et al.,⁴ and Stefano et al.⁵.