Planta (1992)187:405-413 Pl~-_'~ 9 Springer-Verlag1992 Improvements in immunostaining samples embedded in methacrylate: localization of microtubules and other antigens throughout developing organs in plants of diverse taxa Tobias I. Baskin 1., Catherine H. Busby 1, Larry C. Fowke 2, Margaret Saturant 1, and Frank Gubler 3 x Plant Cell Biology, Research School of Biological Sciences, Australian National University, GPO Box 475, Canberra, ACT 2601, Australia 2 Department of Biology, University of Saskatchewan, Saskatoon, Sask. S7N 0W0, Canada 3 Division of Plant Industry, CSIRO, GPO Box 1600, Canberra, ACT 2601, Australia Received 20 November 1991; accepted 1 February 1992 Abstract. Microtubules are important in plant growth and development. Localizing microtubules in sectioned material is advantageous because it allows any tissue of interest to be studied and it permits the positional rela- tions of the cells within the organ to be known. We describe here a method that uses semi-thin (0.5-2 ~tm) sections of material embedded in butyl-methylmethac- rylate, to which 10 mM dithiothreitol was added. After removing the embedding material and using indirect immunofluorescence staining, we obtain clear images of microtubules, actin microfilaments, callose and pulse-fed bromodeoxyuridine. This method works on the root tissues of Arabidopsis thaliana (L.) Heynh, Pinus radiata D. Don, Zamiafurfuracea Ait., Azolla pinnata R. Br. and on sporophytic tissues of Funaria hygrometrica Hedw. In general, most of the cells in the organs studied are suc- cessfully stained. Using this method, we find that inter- phase meristematic cells in all of these species have mi- crotubules not only in the usual cortical array but also throughout their cytoplasm. The presence of the cal- cium chelator ethylene glycol-bis([3-aminoethyl ether)- N,N,N',N'-tetraacetic acid (EGTA) in fixation buffers led to some tissue damage, and did not enhance the preservation of microtubules. The common assumption that EGTA-containing buffers stabilize plant mi- crotubules during fixation appears unwarranted. Key words: Cytoskeleton - Dithiothreitol - Fluorescence microscopy - Immuno-cytochemistry - Microtubules (techniques) *Author for correspondence and reprints. Presentaddress: Division of Biological Sciences,Tucker Hall, University of Missouri, Colum- bia, MO 65211, USA Abbreviations: BrdU= 5-bromodeoxyuridine; DTT=dithiothreitol; EGTA = ethylene glycol-bis(13-aminoethyl ether)-N,N,N',N'- tetraacetic acid Introduction The plant cytoskeleton is a subject of active research. To study its structure, constituent elements must be made visible. The least ambiguous method for this is electron microscopy because it easily resolves structures the size of cytoskeletal elements. However, with electron micro- scopy it is difficult to observe the cytoskeleton through- out large volumes of cytoplasm. This difficulty is sur- mounted by methods based on immunocytochemistry that label the cytoskeleton for observation through the light microscope. These methods cannot always resolve individual cytoskeletal elements because the imaged di- ameter of an element can never be less than the diffrac- tion-based resolution limit, a distance which is more than 10 times the true diameter of a microtubule (Williamson 1991). Nevertheless, relatively large amounts of tissue are readily studied by these methods, and they have been used to gain an invaluable overview of the arrangement of the cytoskeleton (for a recent review, see Lloyd 1987). Because the cell wall blocks antibody penetration, most immunocytochemical studies of the plant cyto- skeleton have been made on wall-less cells, such as endo- sperm or protoplasts, or on isolated cells whose walls have been partially digested enzymatically. However, observa- tion of the cytoskeleton in whole tissue is desirable because of the importance of a cell's position for its development (Barlow and Carr 1984). One approach to overcoming the problem of wall penetration has been to process strips of epidermis that have been micro-surgically removed from the rest of the organ (e.g. Sakaguchi et al. 1988); but this enables study of only the epidermis, and requires non-standard mani- pulation of the tissue. Another approach has been to use sectioned material. To enable sufficient antibody binding for detection of cytoskeletal antigens, the embedding medium must be removed after sectioning. To do this, investigators have used polyethylene-glycol- or wax- embedded sections (Brown et al. 1989) or cryosections