3 D DISTRIBUTION OF CYTOSKELETAL ANTIGENS

我们正在继续努力免疫定位蛋白质， 通过将抗体应用于薄切片， 已经通过冷冻保存技术固定好的样本 并包埋在Lowicryl或LR-White中。 在13种抗体中， 潜在的合作者，只有4个已经产生了良好的控制 具有高于背景的信号的定位， 严格的测试。 我们已经取得了有限的成功， 酵母基因的蛋白质产物 裂殖酵母，但未能定位其他类型的 表位标签。 我们现在正在试验不同的协议， 冷冻取代，在此之前不使用化学交联剂， 低温包埋或替代，但仅添加 醋酸铀酰来增强膜的轮廓。 这些制剂 质量相当高，我们正在进行测试， 切片表面固定剂敏感抗体的反应性 从这种材料上切下来的。 我们能够切割薄带（10- 30 nm） 连续切片使我们能够在更大的范围内收集数据， 表面积与体积比，这对抗原很重要， 以低丰度存在或对于定位于小的 结构.

We are continuing our efforts to immunolocalize proteins in both mammalian cells and in yeast by applying antibodies to thin sections of samples that have been well-fixed by techniques of cryopreservation and embedded in Lowicryl or LR-White. Of 13 antibodies obtained from potential collaborators, only 4 have yielded well-controlled localizations with signals that are above background by statistically rigorous tests. We have had limited success using epitope tagged versions of the protein products of genes from the yeast Schizosaccharomyces pombe, but have failed to localize other types of epitope tags. We are now experimenting with different protocols for freeze-substitution where no chemical crosslinkers are used prior to low temperature embedding or substituting but with only the addition of uranyl acetate to enhance membrane profiles. These preparations are of reasonably high quality, and we are proceeding to test the reactivity of fixative-sensitive antibodies at the surface of sections cut from this material. Our ability to cut ribbons of thin (10-30nm) serial sections is allowing us to gather data at a larger surface-to-volume ratio, which is important for antigens that are present at low abundance or for antigens that localize to small structures.