Immunohistochemical and immunocytochemical studies of gibberellins

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For the immunohistochemistry and immunocytochemistry of gibberellins and GA-binding proteins, monoclonal antibody against GA19 and GA24, and anti-idiotypic antibody against a monoclonal anti-GA4-antibody were prepared. To determine the procedure for fixation of gibberellins, treatments of rapid freezing-freeze substitution in osmate/acetone/dry-ice, and of freeze-drying followed by fixation with gaseous osmate were examined using rice anthers as a plant material, and clear fixation of organelle was observed in the rapid freezing-freeze substitution procedure. The well fixed rice anthers were stained with anti-gibberellin- antibody as the first antibody followed by colloidal gold labeled rabbit anti-mouse IgG. When anti-GA19/24-antibody was used as the first antibody, mitochondria, nuclei (chromatin in particular) and cuticle in epidermal cells of anther, mitochondria, chloroplasts, nuclei (chromatin in particular) and orbiculus in tapetal cells of anther, exine of cell wall, mitochondria and polysaccharide particles in pollen were stained. They were not stained with normal mouse IgG as the first antibody. When anti-GA19/24-antibody preincubated with GA19 was used for immunostaining, nothing was stained, suggesting the specific immunostaining of the above tissues and organelles with anti-GA19/24-antibody. When the anther was stained with anti- GA4-antibody as the first antibody, similar tissues and organelles were stained, but the staining was still observed when stained with the antibody preincubated with GA4 or GA1.