Immunofluorescent localization of pea storage proteins in glycol methacrylate embedded tissue

Improved immunofluorescent techniques have been developed for the high resolution light microscopic localization of intracellular antigens in plant tissue. Thin sections of pea cotyledon tissue which had been fixed in paraformaldehyde and embedded in glycol methacrylate were reacted with mono-specific antibodies to the storage proteins legumin and vicilin. These antibodies were raised in sheep, purified by affinity chromatography and tested by immunoelectrophoresis and immunodiffusion. Using the indirect technique, rhodamine-labeled antibodies permitted specific fluorescent localization of the legumin and vicilin to small (ca. 1 micrometer) cytoplasmic organelles in near mature tissue. Subsequent histochemical staining verified the proteinaceous nature of these organelles. Parameters affecting staining specificity and background fluorescence are discussed.