IMMUNOLABELED MICROTUBULES AND MICROFILAMENTS ARE VISIBLE IN MULTIPLECELL-LAYERS OF RYE ROOT-TIP SECTIONS

A protocol was developed to observe plant microtubules and actin micro filaments in large tissue samples without physical sectioning. Rye (Se cale cereale L. cv. Rymin) root tip pieces from two-day-old seedlings were fixed and processed for immunolabeling. Incubation times of 24-48 h were required to insure adequate penetration of fixatives, antibodi es, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere w ith the resolution of cytoskeletal structures. Microtubules or microfi laments in 5-7 cell layers were visualized using the optical-sectionin g capability of laser scanning confocal microscopy (LSCM) and projecte d as three-dimensional images. The three-dimensional character of the cytoskeletal elements is retained when viewing stained cells of intact tissue, The net-like character of a microfilament array radiating out from a single point in the cytoplasm is maintained when the cells are stained in intact root tip pieces and imaging is accomplished in situ .