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Immunofluorescence and protein A‐gold techniques in localization of plant pathogen antigens in Lowicryl K4M embedded tissue

This paper investigates the use of Lowicryl K4M in the embedding of apple tissue for immunocytochemistry. The localization of the extracellular protease of the apple pathogen, Nectria galligena Bres., in infected apple tissue by immunofluorescence and protein A‐gold immunoelectron microscopy is also described. Infected apple tissue was fixed in 0.5% glutaraldehyde and 4% paraformaldehyde and embedded in Lowicryl K4M at 313 K. The protease was isolated and purified from rotted apple tissue by gel filtration and ion‐exchange chromatography. Monospecific antibodies against the protease were raised in rabbits and purified by protein A‐affinity chromatography. Incubation of apple tissue sections, infected with N. galligena, with the mono‐specific antibody and tetramethylrhodamine isothiocyanate (TRITC)‐conjugate, resulted in specific fluorescence of fungal hyphae and cytoplasm of apple cells. Similar localization of colloidal gold particles over hyphae and host cell cytoplasm was demonstrated employing protein A‐gold immunochemistry. The low temperature embedding resin Lowicryl K4M appears to provide adequate morphological preservation of apple tissue and excellent retention of antigenicity. TRITC conjugates and protein A‐gold may prove useful in immunocytochemical investigations of plant‐pathogen interactions.