के विच्छेदन और धुंधला ड्रोसोफिला लारवल अंडाशय
Summary
कैसे niches और स्टेम कोशिकाओं के विकास के दौरान फार्म व्यावहारिक निहितार्थ के साथ एक महत्वपूर्ण सवाल है. में<em> ड्रोसोफिला</em> अंडाशय, रोगाणु लाइन स्टेम कोशिकाओं और उनके दैहिक niches के लार्वा विकास के दौरान के रूप में. इस वीडियो को दर्शाता है कैसे टुकड़े करना, दाग और देर से तीसरे instar (LL3) से महिला gonads माउंट<em> ड्रोसोफिला</em> लार्वा.
Abstract
Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100μm across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.
Protocol
Discussion
इस वीडियो में एक अलगाव और देर तीसरे instar लार्वा अंडाशय के धुंधला प्रोटोकॉल को दर्शाता है. इस प्रोटोकॉल नियमित रूप से और मज़बूती से प्रदर्शन करने के लिए, निम्नलिखित बातों को ध्यान भुगतान किया जाना चाहिए: </…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
आईएम मैरी क्यूरी पुनः एकीकरण अनुदान द्वारा समर्थित है. यह काम इसराइल विज्ञान कोष अनुदान नहीं द्वारा समर्थित किया गया था. 1146-1108, हेलेन और के Weizmann विज्ञान संस्थान में स्टेम सेल अनुसंधान और Leir चैरिटेबल फाउंडेशन के लिए मार्टिन Kimmel संस्थान द्वारा.
Materials
| Material Name | Tipo | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| NaCl | J.T.Baker | |||
| Kcl | Merck | |||
| CaCl2 | Sigma-Aldrich | |||
| MgCl2 | Merck | |||
| Sucrose | J.T.Baker | |||
| Hepes | Sigma-Aldrich | |||
| PBS | Sigma-Aldrich | |||
| Triton X-100 | Sigma-Aldrich | |||
| Albumin Bovine Fraction V | MP Biomedicals | 160069 | ||
| Dumont biology tweezers 5 dumstar polished | FST Fine Science Tools | 11295-10 | ||
| Nickel plated pin holder | FST Fine Science Tools | 26018-17 | ||
| s.s minutien pins 0.1mm diam, 10mm long | FST Fine Science Tools | 26002-10 | ||
| 9 well plates 85X100 mm, 22mm o.d.x7mm deep | CORNING | 7220-85 | ||
| Stereo Microscope MZ 16.5 with a standard transmitted light base TL ST | Leica | |||
| 6 well plates | Costar | 3516 | ||
| Slides | Menzel Glaser GmbH | 798 | ||
| Cover slips | Corning | 2940-223 | ||
| Mounting media | Vectashield | H-1200 | ||
| Cell strainer | Falcon | FAL352350 | ||
| 1B1 antibody | Developmental Studies Hybridoma Bank | |||
| Anti-Traffic Jam | Laboratory of Dr. Dorothea Godt | |||
| Anti-Vasa | Laboratory of Dr. Ruth Lehmann | |||
| Anti β-Galactosidase | Cappel | |||
| Secondary Antibodies | Jackson ImmunoResearch |
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