本研究为战略研究基金会在民办高校和学术振兴会青年科学家乙津贴为HT我们要感谢大竹德仁编辑的视频片段支持文部科学省支持的项目。

1，准备的苹果平板<ol><li>混合12.5克琼脂，125毫升100％市售苹果汁，12.5克葡萄糖，和375毫升水2 O。为1〜2分钟微波将混合物倒入至3厘米细胞培养皿。贮存于4℃。 </li></ol> 2，遗传交叉<ol><li>使用下列代表的遗传交叉： </li></ol> Or49a-mCD8 :: GFP / CYO x宽1118 3，夹层和染色协议<ol><li>麻醉苍蝇，然后抱着它用钳子剪断飞头垂直。 </li><li>小心地将含天线部分的​​苹果平板。 </li><li>用切细解剖剪刀天线的第三部分。 </li><li>放置90微升的固定溶液（4％多聚甲醛在0.1％的PBST（PBS，0.1％的Triton X-100）），以在玻璃底培养皿的中间。 </li><li>轻轻地转移解剖antennAE用锋利的针直接将固定溶液。如果需要的话，身体浸没触角成使用的针的溶液。 </li><li>孵育40分钟，在室温（RT）。在0.4％的PBST（PBS，0.4％的Triton X-100）洗触角，3X 10分钟每个，保持它们在相同的菜。使用黄色提示，删除和添加PBST解决方案。每次用90微升洗涤液中。 注意：不要将菜成振动筛免疫组化期间。在取出或添加溶液放入盘中，把​​所有的触角到使用针盘的中央，仔细添加或删除从盘边缘的解决方案。 </li><li>与90微升5％正常马血清的0.1％PBST在室温20分钟挡住天线。 </li><li>除去封闭液后，温育在0.1％PBST中含有湿的容器中在4℃下5％马血清48小时初级抗体90μL如先前所描述的天线<s高达&gt; 8。 </li><li>洗触角6X 10分钟0.4％PBST。 </li><li>孵育天线与90微升的二抗在0.1％PBST中含5％马血清48小时，在4℃下使用0.4％PBST洗涤6次10分钟。 </li><li>要安装天线，从培养皿尽可能除去PBST逐步引入两种不同浓度的甘油对触角。首先40％甘油添加到培养皿1-2分钟;然后删除它并添加80％甘油。 </li><li>小心地用黄尖培养皿中提取天线（包括80％的甘油），并放置到幻灯片。轻轻将盖玻片上的顶部和密封盖玻片的边缘用指甲油。该天线现在已经准备好通过荧光显微镜成像。 </li></ol>

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作者什么都没有透露。

果蝇天线，我们描述了解剖简单，易于在实验室环境中进行。以确保成功剥离，有必要利用细边剪刀。而免疫染色的解剖天线，它孵育他们在一个湿气填充容器内，避免抗体溶液的蒸发是很重要的。解剖天线有一种倾向，在溶液中自由浮动。用0.1％的Triton的PBS中固定期间和阻断步骤，将促进溶液中的天线的浸没并以确保更好地染色。使用“玻璃底培养皿”可以减少免疫染色过程中天线的损...

嗅觉系统使用一个巨大的各种气味分子之间进行区分，并随后发送所得到的信息到大脑高级中心。这种输入是用来精确地控制动物的基本行为，如喂养和交配1-6。因为每个嗅觉神经元类型与一组特定的气味，嗅觉受体神经元的关联的多样化（ORN），s是正常嗅觉系统功能7是必不可少的。 果蝇遗传学使我们能够执行涉及与ORN发育和生理功能8-16相关的分子机制单细胞水平的调查。 果蝇触角整装免疫染色，使我们更详细地了解所涉及的嗅觉受体神经元的多样化的分子机制（ORN）秒7。本文中，我们提供了一种简单的方法的全面描述，以交流hieve这一点。

<table border="0" cellpadding="0" cellspacing="0" width="606">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Stemi DV4 dissection microscope</td>
			<td style="width:143px;">Zeiss</td>
			<td style="width:113px;">Stemi DV4</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Glass bottom culture dishes&#160;</td>
			<td style="width:143px;">MatTek corporation</td>
			<td style="width:113px;">P35G-0-10-C</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:235px;">Dissection scissor</td>
			<td style="width:143px;">Fine Science Tools</td>
			<td style="width:113px;">15000-08</td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Rat anti-ELAV</td>
			<td style="width:143px;">Developmental Studies Hybridoma Bank</td>
			<td style="width:113px;">7E8A10</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mouse anti-GFP</td>
			<td style="width:143px;">Invitrogen</td>
			<td style="width:113px;">A11122</td>
			<td style="width:116px;">Dilution 1:400</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rabbit IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">711-225-152</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:235px;">Donkey Anti-Rat IgG</td>
			<td style="width:143px;">Jackson ImmunoResearch Laboratories</td>
			<td style="width:113px;">712-165-150</td>
			<td style="width:116px;">Dilution 1:200</td>
		</tr>
	</tbody>
</table>

whole mount immunolabeling of olfactory receptor neurons in the drosophila antenna

气味分子结合到他们的目标受体的精确和协调的方式。每个受体识别的特定信号并中继该信息到大脑。因此，确定嗅觉信息是如何传递到大脑，修改这两个认知和行为，值得调查。有趣的是，新出现的证据表明，细胞转导和转录因子参与了嗅觉受体神经元的多样化。在这里，我们提供了一个强大的整体安装免疫标记法来测定体内嗅觉受体神经元组织。使用这种方法，我们发现所有的嗅觉受体神经元具有抗ELAV抗体，已知的泛神经标记物和Or49a-mCD8 :: GFP，用抗GFP抗体是NBA神经元中特异性表达嗅觉受体神经元。

确保两个夹层和固定被迅速地进行是在实现成功与此协议的一个关键因素。用细剪刀和镊子也至关重要。染色后，荧光标记的天线进行了共聚焦显微镜下观察。我们通常使用20倍镜头采取1μm的部分。我们标记的NBA使用Or49a-mCD8 :: GFP 7 ORNs并计数的NBA ORNs的野生型天线的数目。该mCD8-GFP报告是细胞膜本地化等的表达见图OR49a ORNs 2展品细胞膜表达GFP。在图2中示出了使用抗GFP抗体和抗ELAV用作泛神?...

Watch this Scientific Journal Video about Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna at JoVE.com

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

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Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

8.9K 次观看.  Doshisha University. Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.