小鸡Statoacoustic神经节和脊髓植在胶原凝胶夹层和文化突起生长实验
Summary
我们演示了如何解剖和文化小鸡E4 statoacoustic节和E6脊髓植。外植体无血清条件下24小时的三维胶原凝胶培养。突起的响应测试与生长因子补充介质与蛋白包被的磁珠。
Abstract
鸡内耳感觉器官外围过程statoacoustic节（SAG）的神经元支配。感觉器官支配取决于轴突的指导线索和生存因素位于沿轨迹的轴突生长和/或在其感觉器官目标2的组合。例如，一个典型的轴突导向信号通路，semaphorin neuropilin，功能产生的干扰misrouting 3奥迪轴突。此外，多种生长因子在内耳的感官指标，包括神经营养因子3（NT – 3）和脑源性神经营养因子（BDNF），表示已在转基因动物的操纵，再次领先misrouting凹陷轴突 4 。同样是这些分子促进体外 5,6和突起生长的小鸡凹陷神经元的生存。
在这里，我们描述和证明在体外的方法，我们目前正在ü唱鸡凹陷突起的可溶性蛋白，其中包括知名morphogens如Wnts，以及促进SAG突起生长和神经元的存活重要的生长因子的响应测试。利用该模型系统，我们希望得出结论分泌的配体可以发挥SAG神经元的存活和突起生长的影响。
凹陷外植体解剖胚胎第4天（E4），并在三维胶原凝胶中培养24小时无血清条件下。首先，突起的响应测试与蛋白培养基中培养的外植体。然后，询问是否分泌配体的点源可以对突起生长的方向影响，外植体共同培养蛋白包被的磁珠和珠本地促进或抑制生长的能力检测。我们还包括一个解剖示范（修改后的协议7）E6脊髓植文化。我们经常使用脊髓植确认生物活性的蛋白质和蛋白质浸泡珠，来验证物种小鸡组织凹陷植相同的培养条件下，交叉反应。这些方便快速筛选分子施加营养（生存）或热带（定向）凹陷神经元的影响，特别是在执行体内研究，在体外实验。此外，这种方法允许单个分子的无血清条件下的测试，具有较高的神经元的存活8。
Protocol
Discussion
我们提出了一个方法来剖析和文化的E4凹陷和E6脊髓植，从小鸡无血清条件下，。目前，这个过程是在我们的实验室研究SAG神经元的存活和突起生长分泌各种配体的影响。本议定书的小说方面，包括使用的无血清培养的外植体浸泡在生长因子的研究凹陷突起生长的影响的珠。珠的方法类似我们已经描述鸡胚脊髓 10 。传统上，珠已被用于研究与经典的轴突导向因子和morphogens。在这里，我们?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由国家卫生部批准RO1DC002756和普渡大学研究基金会。我们感谢与实验的意见和罗德尼帮助数字McPhail多丽丝吴和维泽张。
Materials
| Reagent | Company | Catalogue number | Comment |
| Equipment |
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| Dissection microscope |
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We use a Leica M80 stereomicroscope with brightfield base illumination | |
| Slide warmer | C.S. & E. |
Collagen polymerization | |
| Chicken egg incubator |
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| 37°C/5% CO2 humidified cell culture incubator |
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| Dissection Materials |
|
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| Sylgard© coated petri dish |
|
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| 24-well cell culture plate | Corning |
3526 | |
| #5 Dumont forceps | Fine Science Tools |
11252-30 | |
| #55 Dumont forceps | Fine Science Tools |
11295-51 | |
| Stainless steel dissecting pins | Fine Science Tools |
26002-10 | Embryo pinning, fine dissection |
| Moria perforated spoon | Fine Science Tools |
10370-17 | Embryo harvest/transfer |
| 200 µl wide mouth pipet tips | Dot Scientific Inc |
118-96R | Explant transfer |
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| E4 and E6 chicken eggs |
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| Chick Ringer’s solution |
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Embryo harvest | |
| HBSS | Sigma |
H8264 | SAG dissection |
| L15 medium | Sigma |
L5520 | Spinal cord dissection medium |
| Fetal calf serum | Atlanta Biologicals |
S11150 | Spinal cord dissection medium |
| Vannas Scissors | World Precision Instruments |
501778 | Spinal cord dissection |
|
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| Collagen preparation |
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| Rat-tail collagen Type I | BD Biosciences |
354249 | Explant culture |
| 10X PBS (Sterile) |
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To neutralize collagen | |
| 1N NaOH (Sterile) |
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To neutralize collagen | |
| Distilled water (Sterile) |
|
To neutralize collagen | |
| pH indicator papers (6.0-8.1) | Whatman |
2629-990 | To check collagen pH |
| 15 ml conical tubes | Dot Scientific Inc |
818-PG | |
| 500 µl wide mouth pipet tips | Dot Scientific Inc |
119-R100 | To pipet collagen |
|
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| Explant culture |
|
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| DMEM/F12 medium | Sigma |
D8437 | Explant culture medium |
| ITS+1 | Sigma |
I2521 | Medium supplement |
| Penicillin-Streptomycin | Invitrogen |
15140-122 | Medium supplement |
| CNTF (rat) | Sigma |
C3835 | Medium supplement |
| NT-3 (human) | Sigma |
N1905 | Medium supplement |
| Purified mouse Wnt5a | R&D Systems |
645-WN-010 | |
| Affi-gel Blue gel beads | Bio-Rad |
153-7302 | |
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| Immunohistochemistry |
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| Paraformaldehyde | Sigma |
P6148 | Fixation |
| PBS |
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Rinses | |
| Triton X-100 | Sigma |
T9284 | Blocking solution |
| Sodium azide | Sigma |
S8032 | Blocking solution |
| Calf serum | Invitrogen |
16170-078 | Blocking solution |
| Mouse monoclonal IgG2b anti-β Tubulin 1+II antibody | Sigma |
T8535 | Labels cell bodies and neurites |
| Alexa fluor 488 goat anti –mouse IgG2b antibody | Invitrogen |
A21141 | Detects β Tubulin antibody |
| Teflon micro spatula | VWR |
57949-033 | Release collagen gels from well |
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