解剖和免疫染色成虫盘的<em>果蝇</em
Summary
The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.
Abstract
后期胚胎发育中的果蝇， 果蝇的显著部分，需要在一组囊样结构，称为成虫盘的地方。这些光盘引起成人结构的成年果蝇中发现的高百分比。在这里，我们描述了优化，回收这些光盘并准备分析的抗体，记者转录和蛋白质陷阱的协议。此过程是最适合用于薄的组织像成虫盘，但可以很容易地修改为用较厚的组织使用，如幼虫脑和成年卵巢。书面协议和相应的视频将通过三龄幼虫，组织固定和治疗成虫盘与抗体的夹层引导读者/观众。该协议可以用于解剖成虫盘从年轻的第一和第二龄幼虫为好。这个协议的优点是，它是比较短的，它有可连接优化的高品质保存的解剖组织。另一个优点是，所采用的固定方法可以很好地承认果蝇蛋白质的抗体的数量相当多。根据我们的经验，有极少数不以这个程序很好地敏感的抗体。在这种情况下，补救似乎是使用替代固定的鸡尾酒，同时继续遵循我们提出的解剖步骤和抗体孵育的准则。
Introduction
一个多世纪的果蝇， 果蝇 ，一直是首屈一指的系统来研究发展，行为和生理。发展中的苍蝇可分为两大阶段：胚胎和胚后得多，后者正在发生的范围内的单层上皮称为成虫盘1-3。 成虫盘的图纸首次出版于1864年八月魏斯曼作为他的专着广泛的昆虫开发1份 。这些光盘胚胎发育过程中开始了他们的发展，在幼虫阶段进行构图，生存下来的早期蛹阶段的大规模histolysis，最终导致成年结构被发现在成人中飞往1-14比例很高。在幼虫发育每张光盘公司对于命运，形状和大小的几个关键决策。在第一和第二龄幼虫，光盘的任务是采取主的命运，establis兴隔室的边界，采取了正确的形状和单元格生成15-16的必要数量。在第三龄幼虫和早期预蛹期，成虫盘继续分化和图案化为细胞通过它们的终端命运16。
在果蝇发育生物学的早期历史，成虫盘是在正常发展的情况下，并在有限的情况下，其中的损失或获得功能的突变体是可行的研究几乎完全。利用X射线来诱导允许致死突变有丝分裂重组到在幼虫和成体组织内的细胞克隆进行分析。此方法已被引入转基因方法提高分析损耗和增益的函数在两个幼体和成体组织中的突变。抗体，转录记者和蛋白质陷阱用于说明野生型和突变型组织中的分子景观的数目也是常量antly增长。使用这些分子标记分析损耗和增益的函数的突变体细胞克隆已经变得日益可行获得的如何从他们的野生型表亲发育过程中的突变细胞偏离实时了解。要正确利用这些工具和试剂，关键是要具有可观察，拍照和分析成虫盘高品质的准备。该原稿的目的是提供一种用于眼触角圆盘复合物（ 图1A）的分离和制备的优化协议。它也可以被成功地用于分离各种各样的另外的光盘包括那些引起的翅膀，halteres，T1-T3的腿和生殖器（ 图1B-E）。这个过程，有稍作修改，已用于隔离成虫盘果蝇了近80年。
如上所述，由于大多数的基因中亩表达ltiple发展，在许多组织中的阶段，它往往是不可能的学习效果无效突变对整个眼睛的动物的三龄幼虫期前去世好。四种方法取得了比较发达的组织学习，如视网膜显著更容易处理。第一个是一个否则野生型组织17-19内产生突变体细胞克隆的翻转酶（FLP）/翻转酶的重组目标（FRT）的方法。在此实例中，突变体组织是由缺少可视标记的识别，例如绿色荧光蛋白（GFP），并且可以比周围的野生型组织中的GFP存在（ 图2D）。第二是其中的转基因表达在细胞20的人口的“FLP出”方法。在此实例中的细胞克隆是通过GFP的存在鉴定和比周围的野生型组织的缺乏GFP报告（ 图2E）。三是具有可抑制细胞标记（MARCM）技术，它结合了FLP / FRT突变克隆和FLP出表达系统21元素的马赛克分析。用这种方法的转基因可在细胞，同时突变为单个基因座的群体来表达。像FLP出克隆，MARCM克隆通过GFP的存在鉴定和比周围的野生型组织的缺乏GFP标记（ 图2F）。最后，该基因和RNAi构建体可以特异性启动子-GAL4构建体的控制下，成虫组织中表达。这四种方法都增加学习成虫盘的兴趣，因为突变或过度表达克隆或图案可以直接比较，以相邻的野生型组织。在此过程中描述的方法已经被开发，使研究人员谁研究成体组织中的后胚胎发育的果蝇 ，特别是那些从眼触角光盘衍生，将能够获得高品质的组织进行分析。虽然个别研究人员已经作出轻微的修改，这个过程（其中我们在这里描述）的核心基本上保持不变。由于获得高品质的组织是在成虫盘，我们希望这份书面协议，并伴随视频将作为宝贵的教学资源的研究是至关重要的。
Protocol
Representative Results
Discussion
虽然这个过程在很大程度上集中在分离和后续治疗的眼触角光盘，它是适合于被用于分离和分析的翼，haltere，腿和生殖器光盘（ 图4）。该协议用于分离这些光盘（相对于眼触角光盘）的唯一必需的修改是粗解剖（该协议的第2部分）的方法。第一胸椎腿（T1）对被发现在幼虫的前，可以通过以下的协议眼触角盘隔离来回收。然而，第二胸椎腿（T2）的光盘安装到角质层。以隔离这些光…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们要感谢唐纳德准备和凯文·摩西教授JPK原成虫盘解剖过程。我们也感谢邦妮Weasner的生殖器盘图1B和眼盘图2A，布兰登Weasner为图3中，布鲁明顿果蝇库存中心飞污渍和发展研究杂交瘤细胞银行抗体。 CMS已经由卫生研究院（NIH）的GCMS培训格兰特国家研究院（T32-GM007757），弗兰克·W·普特南研究奖学金，以及罗伯特·布里格斯研究奖学金助学金的支持。 JPK支持由国家眼科研究所资助（R01 EY014863）
Materials
| Name of Material/Equipment | Company | Catalog Number | Comments |
| Sylgard 184 Silicone Elastomer Kit | Dow Corning | 184 SIL ELAST KIT 0.5KG | Used to create base for dissection plate |
| Pryex Glass Petri Dish 150x20mm | Dow Corning | 3160-152CO | Use the cover for dissection plate |
| #5 Dissecting Forceps | Ted Pella | 525 | Forceps must be kept very sharp |
| 9 well watch glass | Vairous Vendors | N/A | Used for fixation of imaginal disc complexes |
| 50ml Erlenmeyer Flask | Various Vendors | N/A | |
| Small Stir Bar | Various Vendors | N/A | Small enough to fit into Erlenmeyer Flask |
| 50ml Conical Tubes | Various Vendors | N/A | |
| 1.5ml Microfuge Tubes | Various Vendors | N/A | Clear or Dark depending upon application |
| Microfuge Rack | Various Vendors | N/A | |
| Benchtop Rotator | Various Vendors | N/A | 100ul volume should not splatter at low setting |
| Paraformaldehyde | Macron Chemicals | 2-26555-1 | Serves as fixative |
| Sodium Phosphate Monobasic | Sigma Chemical Co | S-3139 | Used to make dissection and wash buffers |
| Sodium Phosphate Dibasic | Sigma Chemical Co | 71636 | Used to make dissection and wash buffers |
| Lysine | Acros Organics | 125221000 | Used in the fixative solution |
| Sodium Periodate | Sigma Chemical Co | S-1878 | Used in the fixative solution |
| Triton X-100 | EMD Chemicals | MTX1568-1 | Used to perforate imaginal discs |
| Sodium Hydroxide | EM Science | SX0593-3 | Used to dissolve paraformaldehyde |
| 100% Normal Goat Serum` | Jackson Laboratories | 005-000-121 | Serves as a blocking solution |
| Primary Antibodies | Various Vendors | N/A | Dilute in 10% goat serum as directed by manufacturer |
| Seondary Antibodies | Various Vendors | N/A | Dilute in 10% goat serum as directed by manufacturer |
| Vectashield | Molecular Probes | H-1000 | Prevents bleaching of samples |
| Microscope Slides | Fischer Scientific | 48312-003 | |
| Glass Cover Slips 18x18mm | Fischer Scientific | 12-542A | |
| Kimwipe Tissue | Various Vendors | NA | Prevents Glass slides from adhering to silicone base |
| Panit Brush 000 | Various Vendors | NA | Use to gently lower coverslip on to samples |
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