title: 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.
authors.affiliation: Département de Phytologie, Faculté des Sciences de L'Agriculture et de l'Alimentation, Université Laval, Sainte-Foy, Québec, Canada.
authors.forename: N
authors.initials: N
authors.lastname: Benhamou
authors.forename: S
authors.initials: S
authors.lastname: Noel
authors.forename: J
authors.initials: J
authors.lastname: Grenier
authors.forename: A
authors.initials: A
authors.lastname: Asselin
mesh_terms: D000941:Antigens / Q000032:analysis*; D005110:Extracellular Space / Q000276:immunology; D005404:Fixatives; D008854:Microscopy, Electron; D008872:Microwaves; D010940:Plant Proteins / Q000276:immunology*; D010947:Plants, Toxic; D012680:Sensitivity and Specificity; D014021:Tissue Preservation; D014026:Nicotiana / Q000276:immunology / Q000382:microbiology / Q000648:ultrastructure*; D014027:Tobacco Mosaic Virus / Q000472:pathogenicity
chemical_list: D000941:Antigens; D005404:Fixatives; D010940:Plant Proteins; C053376:pathogenesis-related proteins, plant