Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster
Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster
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DOI：
10.3791/51792
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发表时间：
2014-09-01
影响因子：
1.2
通讯作者：
Kumar, Justin P.
中科院分区：
文献类型：
--
作者：
Spratford, Carrie M.;Kumar, Justin P.
果蝇（Drosophila melanogaster）胚胎后发育的一个重要部分发生在一组称为成虫盘的囊状结构中。这些盘引起在成蝇内发现的高百分比的成蝇结构。在这里，我们描述了一个协议，已被优化，以恢复这些光盘，并准备与抗体，转录报告和蛋白陷阱分析。这种方法最适合像成虫盘这样的薄组织，但也可以很容易地修改用于较厚的组织，如幼虫的大脑和成虫的卵巢。书面协议和随附的视频将指导读者/观众通过解剖三龄幼虫，固定组织，并与抗体治疗成虫盘。该方案可用于解剖成虫盘从年轻的第一和第二龄幼虫以及。该方案的优点是它相对较短，并且已经针对解剖组织的高质量保存进行了优化。另一个优点是，所采用的固定程序与绝大多数识别果蝇蛋白质的抗体一起工作良好。根据我们的经验，有极少数敏感抗体不能很好地与此程序配合使用。在这些情况下，补救措施似乎是使用替代固定鸡尾酒，同时继续遵循我们为解剖步骤和抗体孵育制定的指南。
A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.