Sample preparation for fluorescence imaging of the cytoskeleton in fixed and living plant roots

During the past decade the use of live cytoskeletal probes has increased dramatically due to the introduction of the green fluorescent protein. However, to make full use of these live cell reporters it is necessary to implement simple methods to maintain plant specimens in optimal growing conditions during imaging. To image the cytoskeleton in living Arabidopsis root cells, we rely on a system involving coverslips coated with nutrient supplemented agar where the seeds are directly germinated. This coverslip system can be conveniently transferred to the stage of a confocal microscope with minimal disturbance to the growth of the seedling. Parallel to our live cell imaging approaches, we routinely process fixed plant material via indirect immunofluorescence. For these methods we typically use nonembedded vibratome-sectioned and whole mount permeabilized root tissue. The clearly defined developmental regions of the root provide us with an elegant system to further understand the cytoskeletal basis of plant development.