Microwave energy fixation of plant tissue: an alternative approach that provides excellent preservation of ultrastructure and antigenicity

Abstract

Immunocytochemical techniques are confronted with the problem of obtaining adequate tissue preservation together with retention of protein antigenicity. Various methods, including freeze-drying and freeze-substitution, have been devised to circumvent this problem. In the present study, we report that microwave energy used in combination with low concentrations of glutaraldehyde (0.1%) and paraformaldehyde (2%) preserved the structural integrity of plant tissue and antigenicity of proteins. Tobacco leaf samples fixed in a time as brief as 15-20 s exhibited excellent preservation of fine structures. By contrast, specimens irradiated for shorter (5-10 s) or longer (30-40 s) periods showed poor morphological preservation. Microwave irradiation for 15-20 s was found useful for immobilizing large amounts of soluble antigens. The fast microwave fixation method was successfully used to preserve pathogenesis-related (PR) proteins, which were subsequently localized by a postembedding immunogold procedure. In addition to soluble antigens, cellulose subunits and pectic substances, two major plant cell wall components, were found to be highly preserved in microwave-irradiated tobacco plant tissue. The present study demonstrates that microwave fixation of plant tissue is a simple and inexpensive method that is easy to perform with commercially available microwave ovens. The incubation time for fixation is reduced from 2 h to 15-20 s without loss of fine structural details. This method will undoubtedly acquire increasing applicability and relevance in plant biology.