IMMUNOLABELED MICROTUBULES AND MICROFILAMENTS ARE VISIBLE IN MULTIPLE CELL LAYERS OF RYE ROOT TIP SECTIONS

摘要

A protocol was developed to observe plant microtubules and actin microfilaments in large tissue samples without physical sectioning. Rye (Secale 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, antibodies, and washing buffers. Clearing of the tissue with methyl salicylate reduced background auto-fluorescence that would otherwise interfere with the resolution of cytoskeletal structures. Microtubules or microfilaments in 5-7 cell layers were visualized using the optical-sectioning capability of laser scanning confocal microscopy (LSCM) and projected 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. [References: 28]