Immunofluorescent Staining of Microtubules in Plant Tissues: Improved Embedding and Sectioning Techniques using Polyethylene Glycol (PEG) and Steedman's Wax

Methods for the indirect immunofluorescent staining of microtubules in embedded and sectioned plant tissues are described and compared. Root tips of Vicia faba, Saccharum officinale, Allium cepa, and root nodules of Glycine max were fixed using conventional methods, embedded in polyethylene glycol or Steedman's wax, sectioned with a glass knife on a rotary microtome, and dewaxed in water or alcohol. The addition of dithiothreitol (DTT), dehydrating at low temperatures and reducing the infiltrations times were found to reduce background fluorescence in Allium cepa. Steedman's wax yields a block that is similar to paraffin and is easier to section than PEG. Routine methods for indirect immunofluorescence were used to stain sections for tubulin/microtubules. The major microtubule arrays of mitotic cells are illustrated in this paper. The principal advantage of this technique is the preservation of cell‐to‐cell continuity in multicellular tissues. This method provides a much needed technique for the study of the cytoskeleton during growth and differentiation of plant tissues.