Accumulation of callose (β-1,3 glucans) at the plasmodesmata (PD) neck region dynamically regulates symplastic intercellular transport. Here we describe a 2-3-day immuno-labelling protocol to determine callose levels in the cell wall region at PD. The method relies on exposure of internal cell walls by hand-sectioning of the sample and digestion of the cell wall with enzymes in order to improve antibody penetration to deep tissue layers. By using this protocol, combined with high-resolution confocal imaging, we successfully detected PD-associated callose in Arabidopsis root apical meristem, vascular tissue, and developing lateral root primordia.