Rapid fixation and embedding method for immunocytochemical studies of tomato spotted wilt tospovirus (TSWV) in plant and insect tissues

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Abstract
A new rapid fixation and embedding technique using microwave energy was evaluated for immunolabelling and examination of ultrastructure of plant and insect cells. Tissues in gluteraldehyde‐paraformaldehyde were fixed for fifteen seconds in a microwave at 100% power, and dehydrated. Microwave energy was then used to polymerize the London Resin White (LR White) acrylic resin during the embedding process. Embedded specimens were then thin sectioned (90 nm) and treated with anti‐tomato spotted wilt tospovirus (TSWV) antiserum followed by protein A‐gold label, or antisera against a TSWV encoded nonstructural protein followed by goat anti‐rabbit gold label. Using this technique, structural and nonstructural proteins of TSWV were readily detected and specifically labelled in cells of the insect vector, the western flower thrips, Frankliniella occidentalis (Pergande), and in infected cells of the plant species, Emilia sonchifolia L.