Katoh K, Ichikawa H, Ishikawa H
Department of Anatomy, Gunma University School of Medicine, Japan.
J Electron Microsc (Tokyo). 1991 Feb;40(1):70-5.
Various specimen preparations for thin-section electron microscopy were tested to better preserve and visualize actin filaments in the cortex of the early embryos of Drosophila melanogaster. When embryos were treated with phalloidin prior to fixation, many actin filaments were observed in their cortex comparable to the staining with fluorescently labeled phalloidin in light microscopy. Then we used various fixatives containing phalloidin. As far as we examined, actin filaments were best preserved in the specimen fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M sodium phosphate buffer or in 0.1 M PIPES buffer (1 mM EGTA and 1 mM MgCl2) containing 10 microM phalloidin and 0.1% saponin. When embryos were glycerinated and then treated with tropomyosin before fixation, actin filaments were well visualized as thicker, uniform-sized filaments, though the number of filaments decreased probably owing to glycerination. This suggests that, like heavy meromyosin and its subfragment-1, this protein may protect actin filaments from being disrupted by chemical fixation. Using these improved fixation procedures, we successfully examined the distribution of actin filaments in the Drosophila embryo cortex during cellularization. These methods may be applicable to stabilize labile actin filaments in other types of cells.
为了更好地保存和观察黑腹果蝇早期胚胎皮层中的肌动蛋白丝，我们测试了多种用于超薄切片电子显微镜的标本制备方法。当胚胎在固定前用鬼笔环肽处理时，在其皮层中观察到许多肌动蛋白丝，这与光学显微镜下用荧光标记的鬼笔环肽染色的情况相当。然后我们使用了含有鬼笔环肽的各种固定剂。据我们检查，在用含有10微摩尔鬼笔环肽和0.1%皂素的0.1M磷酸钠缓冲液或0.1M PIPES缓冲液（1mM乙二醇双四乙酸和1mM氯化镁）中的2.5%戊二醛、2%多聚甲醛固定的标本中，肌动蛋白丝保存得最好。当胚胎用甘油处理，然后在固定前用原肌球蛋白处理时，肌动蛋白丝能很好地显示为更粗、大小均匀的丝，尽管由于甘油处理，丝的数量可能减少。这表明，与重酶解肌球蛋白及其亚片段-1一样，这种蛋白质可能保护肌动蛋白丝不被化学固定破坏。使用这些改进的固定程序，我们成功地研究了细胞化过程中果蝇胚胎皮层中肌动蛋白丝的分布。这些方法可能适用于稳定其他类型细胞中不稳定的肌动蛋白丝。
