Immunofluorescence Analysis of Membrane-Associated Proteins for Clathrin-Mediated Endocytosis in Plant Root Cells

The root is an ideal model system for studying subcellular localization and dynamic trafficking of important membrane-associated proteins in plants. Immunofluorescence analysis is necessary to reveal subcellular localization and intracellular trafficking of endogenous proteins as epitope tags or fluorescent proteins may cause mislocation of fusion proteins. Here, we describe a rapid and reliable immunodetection protocol for whole-mount in situ localization of membrane-associated proteins involved in clathrin-mediated endocytosis (CME) in Arabidopsis root cells. The whole procedure includes five basic steps: tissue fixation, tissue permeation, blocking, primary antibody incubation, and secondary antibody incubation.