3 D DISTRIBUTION OF CYTOSKELETAL ANTIGENS

我们正在继续努力对这两种蛋白进行免疫定位。 哺乳动物细胞和酵母细胞通过将抗体应用于薄片 已经通过超低温保存技术很好地固定的样本 并嵌入Lowicryl或LR-White。在13种抗体中，从 潜在的合作者，只有4个得到了很好的控制 利用统计方法对背景以上信号进行定位 严格的测试。我们利用标记的表位取得了有限的成功 酵母基因的蛋白质产物的不同版本 裂殖酵母，但未能本地化其他类型的 表位标签。我们现在正在尝试使用不同的协议来 冷冻置换，在此之前不使用化学交联剂 低温包埋或替代，但仅加成 使用醋酸铀酰来增强膜的轮廓。这些准备工作 具有相当高的质量，我们正在进行测试 固定剂敏感抗体在切片表面的反应性 从这块料子上剪下来的。我们切割薄(10-30 nm)丝带的能力 连载部分使我们能够在更大的 表面积与体积的比率，这对抗原来说很重要 以低丰度存在或针对定位于小分子的抗原 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.