Summary
Abstract
Introduction
Protocol
Representative Results
Discussion
Acknowledgements
Materials
References
Developmental Biology
果蝇卵巢是研究干细胞小生境发育的优良模型系统。虽然已经公布了解剖幼虫和成人卵巢的方法, 蛹卵巢解剖需要不同的技术, 没有详细发表。在这里, 我们概述了解剖, 染色和安装蛹卵巢的协议。
与成年的果蝇卵巢不同, 蛹卵巢由于其体积小、半透明和在蛹的情况下被包裹而难以进入和检查。解剖蛹卵巢的挑战还在于它们在蛹中的物理位置: 卵巢被蛹腹部内的脂肪体细胞所包围, 必须除去这些脂肪细胞, 以允许适当的抗体染色。为了克服这些挑战, 本协议利用自定义的巴斯德滴管从蛹腹部提取脂肪体细胞。此外, 在染色过程中使用腔内 coverglass 代替离心管, 以提高蛹的可见度。然而, 尽管本议定书使用的工具有这些和其他优点, 但由于蛹卵巢体积小, 成功地执行这些技术可能还需要几天的实践。本议定书所概述的技术可应用于蛹发育不同阶段的卵巢分析的时间过程实验。
干细胞研究使用果蝇卵巢已广泛扩展自第一个文件的干细胞利基1,2,3,4。随着沿袭追踪遗传工具的发展,果蝇卵巢解剖通常被用来研究干细胞的谱系和信号通路, 调节干细胞的维持、增殖和命运。有关这些信号通路的知识可能会对源于异常干细胞活动的癌症的潜在病因产生洞察力5,6,7。最近还显示, 在果蝇卵巢中, 称为卵泡干细胞 (开办) 的体细胞干细胞在其组织的许多方面都与哺乳动物的肠道干细胞有很大的相似性, 这些细胞有8。因此,果蝇卵巢是研究干细胞行为的一个非常有用的模型系统。
幼虫和成人卵巢为早期干细胞的发育和生态位的最终干细胞组织提供了线索, 蛹卵巢是生殖和体细胞重组和建立其身份的中间结构。9,10. 虽然有几项研....
Name | Company | Catalog Number | Comments |
Dumont #5 Forceps, biology | Fine Scientific Tools | 11252-20 | |
Nunc Lab-Tek Chambered Coverglass | Thermo Fisher Scientific | 155383 | |
Dissection microscope | Nikon | SMZ-10A | |
Confocal Microscope | Carl Zeiss | LSM 700 | |
Analysis software | Carl Zeiss | Zen | |
9 Depression Glass Spot Plates | Pyrex | 7220-85 | |
Pasteur pipet | Fisher Scientific | 13-678-6B | |
Pasteur pipet bulb | Various vendors | ||
Bunsen burner | Various vendors | ||
Fisherfinest Premium Frosted Microscope Slides | Thermo Fisher Scientific | 12-544-2 | |
22 x 22 mm glass coverslips No 1 | VWR | 48366-067 | |
Dapi Fluoromount-G | SouthernBiotech | 0100-20 | |
Double-sided tape | Scotch | ||
Nutator | Clay Adams | ||
Fine brush #0, #3-#5 | Various vendors | ||
Gilson Pipetman Starter Kit | Thomas Scientific | F167300 | Contains p20, p200, p1000 pipettors |
16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | Dilute to 4% paraformaldehyde in 1x PBS |
Triton | Sigma-Aldrich | 9002-93-1 | |
10x PBS | Ambion | AM9624 | Dilute to 1x PBS |
Normal Goat Serum | Jackson ImmunoResearch | 5000121 | Dilute to 10% normal goat serum in PBST with 0.5% Triton concentration |
Primary antibodies (in protocol: 7G10 anti-Fasciclin III diluted 1:250, rabbit anti-phosphohistone H3 diluted 1:1000) | Various vendors (in protocol: Developmental Studies Hybridoma Bank, Millipore) | Dilute in PBST with 0.5% Triton concentration | |
Secondary antibodies (in protocol: Alexa-546, FITC-conjugated anti-rabbit serum) | Various vendors (in protocol: Molecular Probes, Jackson ImmunoResearch Laboratories, Inc.) | Dilute in PBST with 0.5% Triton concentration | |
Fly vials | Denville Scientific | V9406 | |
Cotton Balls, For Wide Vials | Genesee Scientific | 51-102W | |
Yeast, Bakers Dried Active | MP Biomedicals | 101400 | |
Fly food | Produced in laboratory | Mixture of water, brewer's yeast, cornmeal, molasses, agar, EtOH, penicillin, methyl 4-hydrobenzoate, and propionic acid | |
Male and female Drosophila flies (genotype used in protocol: yw; P[Fz3-RFP, w+]/TM2) | Bloomington Drosophila Stock Center |
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