Freeze shattering: a simple and effective method for permeabilizing higher plant cell walls G. O. WASTENEYS,*† J. WILLINGALE-THEUNE† & D. MENZEL† *Plant Cell Biology Group, Research School of Biological Sciences, The Australian National University, GPO Box 475, Canberra, ACT 2601, Australia †Max-Planck-Institut fu ¨r Zellbiologie, Rosenhof, Ladenburg, D-68526 Germany Key words. Actin filaments, Allium, Arabidopsis, confocal laser scanning microscopy, immunofluorescence, microtubules, Tradescantia. Summary This article describes a practical technique for permeabiliza- tion of higher plant cell walls, which is usually one of the first steps required for immunolocalization of cellular components (and other cytological methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It replaces the use of wall degrading enzymes or the need to employ laborious sectioning or other mechanical means for providing access of probes to cells. Freeze-shattering retains the integrity of whole tissues and cells surprisingly well and thus is especially useful when used in conjunction with confocal laser scanning microscopy for recording the three- dimensional arrangement of cytoskeletal elements in relation to cell shape. In this article, we demonstrate the effectiveness of this technique for anti-tubulin and anti- actin immunofluorescence and for rhodamine phalloidin labelling of the cytoskeleton in various higher plant tissues including onion root tip and bulb scale epidermis, Tradescantia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells. Introduction The study of plant cell structure has profited from cytochemical procedures in which highly specific probes are introduced into fixed cells to reveal the distribution of cellular components. Unfortunately, the plant cell wall excludes molecules larger than 4 nm so strategies must be devised to get bulky globulins and other probes across the cell wall barrier. The use of immunofluorescence in plant cytology was first described in 1970 with the localization of cell wall antigens in pollen grains (Knox, 1970) and proteins in cotyledons (Graham & Gunning, 1970). In these cases, the localization of extracellular antigens (Knox, 1970) and the use of hand sectioning for gross localization of cytoplasmic constituents (Graham & Gunning, 1970) belied the relative difficulty that was to be encountered in using immunofluorescence as a routine procedure for plant tissues. Although plant protoplasts (Lloyd et al., 1980; Van der Valk et al., 1980) and naturally naked plant cells (Franke et al., 1977) can be processed as easily as animal cell types, penetrating the cell wall and maintaining the integrity of cell contents in most plant cells is problematic. Two principal approaches have been employed for permeabilizing plant cell walls. The first approach uses wall-degrading enzymes that break down component polysaccharides and is effective for organs such as root tips (Wick et al., 1981). It is common, however, for enzyme treatments to break up complex tissues into their compo- nent cells so that the integrity of the tissue or organ is no longer discernible. Furthermore, enzymatic treatments are usually inadequate for permeabilizing cells that are protected by a waxy cuticle. Larsen & Wolniak (1994) have recently developed a method that uses cutinase incubation prior to fixation in order to remove the cuticle of Tradescantia stamen hair cells but this method requires incubation of living cells, a requirement almost certainly suboptimal for preservation of the cortical cytoskeleton (Lee et al., 1989). Other approaches to plant cell wall permeabilization rely on physical opening of cells (Menzel & Schliwa, 1986). Such methods have proven effective when enzyme treatments are unsuccessful or do not preserve the integrity of the tissue. In the exceptional case of giant internodal cells of characean algae, vacuolar perfusion permits easy permeabilization of cells for antibody access (Williamson & Toh, 1979) but even this convenient method is not optimal for preservation of elements nearest the plasma membrane (Wasteneys et al., 1996). Material can also be embedded and sectioned to expose the cells to antibodies after subsequent removal of Journal of Microscopy, Vol. 188, Pt 1, October 1997, pp. 51–61. Received 10 December 1996; accepted 23 March 1997 51  1997 The Royal Microscopical Society Correspondence to: G. O. Wasteneys, Tel:+61 (6) 249 3952; fax: +61 (6) 249 4331; email: geoff.wasteneys@anu.edu.au