Immunolabeling Sieve Element Cell Walls with the LM26 Antibody

Sieve elements in the phloem transport carbon and small molecules, such as RNA and phytohormones, throughout the plant body. Understanding the physical dimensions of sieve elements and phloem tissue is thus crucial for predicting how much carbon can be moved at any given time. Quantification of sieve element diameters and areas has previously been performed using transmission electron microscopy, scanning electron microscopy, and light microscopy, but sieve element identification is difficult because the phloem is a heterogeneous tissue. The recently identified LM26 antibody labels a pectin in the sieve element cell wall, allowing the identification of sieve elements and the measurement of their properties, such as diameter, relatively quickly and the quantification of their number in cross sections using image analysis software. Here, we describe methods for immunolabelling sieve elements in fresh or fixed tissue embedded in polyethylene glycol or methacrylate. The protocol is broadly adaptable to various fixation and sectioning methods, provided they do not alter the structure of pectins in the cell wall.