Immunohistochemical and immunocytochemical studies of gibberellins

赤霉素和ga结合蛋白的免疫组织化学和免疫细胞化学制备了针对GA19和GA24的单克隆抗体和针对ga4单克隆抗体的抗独特型抗体。为了确定赤霉素的固定过程，以水稻花药为原料，研究了在浸膏/丙酮/干冰中快速冷冻-冷冻替代处理，以及在浸膏/丙酮/干冰中冷冻干燥后再用气体浸膏固定的处理，在快速冷冻-冷冻替代过程中观察到细胞器的明显固定。将固定好的水稻花药用抗赤霉素抗体作为第一抗体进行染色，然后用胶体金标记兔抗小鼠IgG进行染色。以抗ga19 /24抗体为第一抗体，对花药表皮细胞的线粒体、细胞核（特别是染色质）和角质层，花药绒毡层细胞的线粒体、叶绿体、细胞核（特别是染色质）和圆形细胞、细胞壁外壁、花粉中的线粒体和多糖颗粒进行染色。未以正常小鼠IgG作为第一抗体进行染色。用GA19预孵育的抗GA19/24抗体进行免疫染色，未见任何染色，提示抗GA19/24抗体对上述组织和细胞器进行了特异性免疫染色。以抗GA4抗体为第一抗体对花药进行染色时，可染色到类似的组织和细胞器，但与GA4或GA1预孵育的抗体染色时，仍可观察到染色。

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.