FREEZE SHATTERING - A SIMPLE AND EFFECTIVE METHOD FOR PERMEABILIZING HIGHER-PLANT CELL-WALLS

This article describes a practical technique for permeabilization of h igher plant cell walls, which is usually one of the first steps requir ed for immunolocalization of cellular components (and other cytologica l methods) in plant cell studies. Our strategy involves shattering the walls of cells while the tissues are frozen in liquid nitrogen. It re places the use of wall degrading enzymes or the need to employ laborio us sectioning or other mechanical means for providing access of probes to cells, Freeze-shattering retains the integrity of whole tissues an d cells surprisingly well and thus is especially useful when used in c onjunction with confocal laser scanning microscopy for recording the t hree-dimensional arrangement of cytoskeletal elements in relation to c ell shape, In this article, we demonstrate the effectiveness of this t echnique for anti-tubulin and antiactin immunofluorescence and for rho damine phalloidin labelling of the cytoskeleton in various higher plan t tissues including onion root tip and bulb scale epidermis, Tradescan tia stamen hairs and Arabidopsis leaf epidermis and mesophyll cells.