Poster: Emerging technologies

Abs # 914: Development of microwave-assisted specimen preparation methods for electron microscopic immunolocalization in whole Arabidopsis tissues

Presenter:	Avci, Utku , uavci@unity.ncsu.edu
Authors	Avci, Utku  (A)   Grimson, Mark J (B)   Haigler, Candace H. (A)
Affiliations:	(A): North Carolina State University (B): Texas Tech University

A full understanding of protein function implies a need to understand protein location within organelles, which is typically below the level of resolution of fluorescence and confocal microscopy. Preparation of specimens for immunolocalization in the electron microscope is time-consuming, the degree of ultrastructural preservation is often poor, and proteins have ample opportunity to move in traditional preparative protocols. Microwave-assisted procedures that minimize these problems have been described (1), but they are not widely used by plant scientists. Microwaves (MWs) are a form of non-ionizing radiation with an output frequency of 2.45 GHz (billion cycles/second) corresponding to a wavelength of 12.2 cm. Microwaves provided by a programmable, laboratory grade, microwave (e.g. Pelco Biowave 34700, Ted Pella, Redding, CA) can greatly enhance the speed of fixative penetration and action and ultrastructural quality (1). We are testing and optimizing existing protocols using seedlings of Arabidopsis thaliana (Columbia) and 4% formaldehyde as fixative, which should retain protein antigenicity. With a protocol that requires only one hour before intact seedling roots are fully infiltrated in 100% resin, excellent sub-cellular ultrastructural preservation as observed in the electron microscope has been achieved in all cells of the Arabidopsis seedling root. Testing of immunolabeling is in progress and will be reported. (1) Russin WA, Trivett CL (2001) Vacuum-microwave combination for processing plant tissue for electron microscopy. In: Microwave Techniques and Protocols, R.T. Giberson and R. S. Demaree Jr, eds., Humana Press: Totowa, NJ, p. 25.