| 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%) preserves 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. |
