Methanol-acetone fixation of Drosophila testes.
Methanol-acetone fixation of Drosophila testes.
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DOI：
10.1101/pdb.prot065763
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发表时间：
2011-10-01
影响因子：
--
通讯作者：
Gatti, Maurizio
中科院分区：
文献类型：
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作者：
Bonaccorsi, Silvia;Giansanti, Maria G;Gatti, Maurizio
该协议描述了两种用于果蝇睾丸甲醇-丙酮固定的技术。第一种方法可以很好地保存细胞形态。通过相差光学观察的固定细胞显示了活体材料中可以看到的大部分结构细节。这样可以分析未染色的固定制剂并选择最适合免疫染色的制剂。值得注意的是，Y 环在活体制剂中通常是微弱且不稳定的，但在这种类型的固定后变得清晰可见。此外，这种固定方法可以很好地保存微管，用于抗微管蛋白抗体的免疫染色。该技术的主要缺点是染色体结构保存较差。在大多数情况下，染色体不表现出独特的形态，并且倾向于合并成一团或多团染色质。此处描述的第二种甲醇-丙酮固定技术已被证明特别适用于 γ-微管蛋白和中心体蛋白免疫染色。其产生的制剂具有与第一种方法获得的制剂相同的特性。
This protocol describes two techniques for methanol-acetone fixation of Drosophila melanogaster testis squashes. The first method results in very good preservation of cell morphology. Fixed cells viewed by phase-contrast optics show most of the structural details that can be seen in live material. This allows analysis of unstained fixed preparations and selection of the most suitable ones for immunostaining. Remarkably, the Y loops, which are usually faint and labile in living preparations, become clearly apparent after this type of fixation. Moreover, this fixation method results in excellent microtubule preservation for immunostaining with antitubulin antibodies. The main disadvantage of this technique is poor preservation of chromosome structure. In most instances, the chromosomes do not show a distinct morphology and tend to coalesce into one or more masses of chromatin. The second technique for methanol-acetone fixation described here has proved to be particularly suitable for gamma-tubulin and centrosomin immunostaining. It results in preparations having the same characteristics as those obtained with the first method.