作者要感谢迈克尔·安德森建立在李某实验室研究与精子从卡伦海尔斯专家建议这些接受的方法。 H.小田和Y秋山织田慷慨提供的γ-微管蛋白GFP飞股票。这项工作是由美国国立卫生研究院的R01授予LAL（GM074044）的支持。

1。睾丸解剖<ol><li>使用CO 2流麻醉苍蝇在瓶子或小瓶中，并转移到飞盘。 </li><li>使用小画笔在解剖显微镜下排序苍蝇，并收集所需的基因型果蝇雄性适当数量（取决于实验）。年轻男性（0-2日龄），非常适合在整个精子发生的早期阶段（ 如精原细胞，精母细胞和早期减数分裂后的精子细胞）研究细胞，而年龄稍大的男性（2-5日龄）是理想的研究细胞在精子发生过程的最后阶段（尤其是成熟的精子）。 </li><li>使用镊子从各飞取下翅膀（防止苍蝇剥离时漂浮在液体）。 </li><li>加〜500毫升磷酸盐缓冲盐水（130 mM氯化钠，7毫米的Na 2 HPO 4，3mM的的NaH 2 PO 4; PBS）中的一个降低到了聚硅氧烷涂覆的解剖盘上一个黑色的背景。其它水溶液已被成功地用于睾丸切除3。 </li><li>点钳朝飞前，抓住它的胸部，并沉浸在PBS的下降。同时通过解剖显微镜观看，请使用另一双钳抓住并拉动外生殖器（位于腹腹部的后端深褐色结构）向后，直到它从腹部分离。在大多数情况下，睾丸，精囊，和附腺将被从腹部随着外生殖器取出，若否，插入一对镊子进入腹部和梳理出睾丸。 </li><li>睾丸分开使用两对PBS的下降钳（ 图2），附属性腺及外生殖器。野生型睾丸很容易从周围的白色组织中的黄颜色区分。立即进行第2步。 </li><li>从pharate男性（ 我睾丸隔离。辰蛹壳括起来），一个额外的步骤必须先执行，涉及从蛹壳清除飞;这一步之前已在别处31描述。进行清扫作为成人睾丸开始在步骤1.2。 </li><li>从幼虫男性睾丸分离，分离果蝇幼虫卵巢32执行协议的修改。简言之，男性幼虫可从女性的幼虫由一对嵌在脂肪体的后部第三大而明确，椭圆形结构（幼虫睾丸）存在区别。为了隔离幼虫睾丸，部分一气之下打开雄性幼虫从腹部所描述的幼虫卵巢的隔离，隔离和睾丸周围脂肪体。立即进行第2步本文所述的协议;睾丸以后可以从脂肪体安装（步骤2.3或3.14）所描述的卵巢32之前，为了去除。 </li></ol><ol><li>使用镊子轻轻放置于4-5毫升的PBS在方形盖玻片下降2-3对睾丸。需要注意的是睾丸数目与PBS体积之比，可能需要进行调整：液体过多会防止细胞被挤压时扩频正确的，而过少的液体将导致细胞爆裂压扁时。可选：使用硅化盖玻片，尽量减少组织坚持以盖玻片在步骤3.2。 </li><li>使用镊子撕开每个睾丸在适当的位置，以最大化所需的生殖系细胞类型在编写存在（请注意，睾丸会多出从撕开区域到幻灯片中的挤压过程中的内容步。）为丰富的精原细胞和精母细胞，拆毗邻其顶端尖（级别1， 图2B）的睾丸。以丰富的精母细胞和精子细胞，撕裂在POS打开睾丸银行足球比赛稍有基础，以1级（2级， 图2B）。为了丰富更成熟的生殖细胞，撕开睾丸接近那里的曲率开始（一级3， 图2B）。 </li><li>轻轻地放在显微镜载玻片在盖玻片壁球睾丸，不要手动施加压力作为单独盖玻片的重量足以获得适当压扁的样品。尽量避免捕获气泡。可选：使用聚-L-赖氨酸包被的载玻片，以促进组织的粘附，以在步骤3.2滑动。 </li><li>立即使用制剂（理想的范围内制备的15分钟）通过相差显微镜观察活细胞;为转基因果蝇与睾丸的荧光标记蛋白的表达，活细胞可以通过荧光显微术在这个步骤检查。另外，进行固定和抗体染色（协议3）。 </li><li>轻轻地用清洁瓦特从盖玻片下灯芯任何多余的液体IPE以便编写的扁平化，直至生殖细胞显然是关注的焦点。 </li></ol> 3。甲醛固定和抗体染色<ol><li>急速冷冻每片含挤压睾丸（从协议2）使用一对金属钳简单浸在液氮中（直到液氮停止冒泡）的幻灯片。 </li><li>使用刀片立即取出盖玻片。 </li><li>使用金​​属钳到载玻片转移至充满了冰冷的95％乙醇中的预冷的载玻片架（分光级，无甲醇）。储存在-20℃下进行10分钟。 </li><li>使用金​​属钳到载玻片转移到填充有4％多聚甲醛的PBS中加0.1％的Triton X-100的PBS（PBS-T）的载玻片架。储存在室温下为7分钟。 </li><li>用金属钳，以幻灯片转移到充满PBS载玻片架。在PBS中5分钟，在室温下洗涤​​载玻片。重复1X。通过丢弃的解决方案在载玻片架（执行所有清洗<em&gt; IE浏览器。浇出来），并用新鲜的解决方案取代。 </li><li>弃去PBS中并浸泡在PBS-T中的幻灯片30分钟，在室温下透化细胞膜。 </li><li>在PBS中5分钟，在室温下洗涤​​载玻片。重复2倍。 </li><li>阻断步（可选）：载玻片浸泡在PBS中加1％的BSA在室温下45分钟。 </li><li>使用疏水屏障用笔画出周围压扁组织（肉眼很容易看到），以限制该抗体溶液（每步3.9和3.11加）滑了一圈。组织应保持湿润在任何时候都同时进行免疫染色。 </li><li>加30〜40毫升的第一抗体（稀释于PBS-T中，1:400至1:50，这取决于抗体），以组织的圆内。如果进行拦截，稀释的一抗在PBS-T +1％BSA中。抗γ-微管蛋白抗体（在图4中的中心体的染色）以1:100稀释。孵育在潮湿，黑暗的室内（ 例如 ，封闭的塑料盒用湿纸巾）2小时，在室温或4℃过夜°C。 </li><li>在PBS中5分钟，在室温下洗涤​​3次滑动。如果进行阻断，在PBS-T洗涤两次，一次在PBS（5分钟，在室温下每个）。 </li><li>加30-40毫升的荧光团标记的二抗（稀释在PBS中1:400）到组织和孵育在黑暗中1-2小时，在室温下进行。 </li><li>在PBS中5分钟，在室温下洗涤​​载玻片。重复2倍。 </li><li>加30-40毫升的DAPI溶液（0.2 mg / ml的PBS）中的圆圈内的组织。 </li><li>轻轻将盖玻片在组织，同时注意避免诱捕气泡。如果气泡应该出现，小心周围的盖玻片移动，而不会破坏壁球，直到气泡从盖玻片的两侧逃跑。 </li><li>使用清洁擦拭从幻灯片的边缘吸干多余的DAPI。 </li><li>用透明指甲油密封盖玻片的幻灯片。 </li><li>使用这种制剂在未来的3-4小时内，荧光显微镜，查看免疫染色细胞。对于较长期存储的幻灯片（高达至少几个星期），可以使用甘油为基础的硬安装介质用DAPI和存储载玻片在-20˚C。 </li><li>或者，固定样品在甲醇代替甲醛（取决于抗原）。通过3.2步完成整个协议后，浸泡挤压睾丸幻灯片甲醇10分钟，在-20°C和继续步骤3.5起。 </li></ol>

<ol>
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H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118, 401-415 (1993).</li><li>White-Cooper, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+cell+type+and+stage-specific+ectopic+gene+expression+and+RNAi+induction+in+the+Drosophila+testis.">Tissue cell type and stage-specific ectopic gene expression and RNAi induction in the Drosophila testis.</a> Spermatogenesis. 2, 11-22 (2012).</li><li>Rebollo, E., Llamazares, S., Reina, J., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+noncentrosomal+microtubules+to+spindle+assembly+in+Drosophila+spermatocytes.">Contribution of noncentrosomal microtubules to spindle assembly in Drosophila spermatocytes.</a> PLoS Biol. 2, (2004).</li><li>Cheng, J., Hunt, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-lapse+live+imaging+of+stem+cells+in+Drosophila+testis.">Time-lapse live imaging of stem cells in Drosophila testis.</a> Curr. Protoc. Stem Cell. Biol.. 2, 10-1002 (2009).</li><li>Sheng, X. 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作者宣称，他们有没有竞争的财务权益。

虽然野生型果蝇的睾丸可以很容易由于其黄色（相邻近的白色组织）鉴定， 白色突变果蝇的睾丸是白色的，因此可以偶尔与肠道混淆。大多数转基因株系，这是典型的在一个白色的背景，也有白色的睾丸，因为在P -元素发现的迷你白基因不提倡色素积聚在睾丸。当果蝇睾丸不能用颜色加以区分，其它易于识别的功能包括对12的螺旋纹和发生。请注意，一?...

果蝇睾丸是许多生物过程，包括干细胞的调节，减数分裂和精子发育13-18的研究的理想模型系统。在精母细胞和其减数分裂主轴都很大，进行细胞学分析，因此方便，而且精子发生过程轻松细胞周期检查点方便突变在细胞周期基因的研究。不同类型的细胞可以在有序进展可以观察到沿睾丸的长度，并在精子发生任何干扰可以导致改变这个布局。这些功能结合果蝇的遗传工具提供了便利21-23精子的突变分析。 果蝇精子发生的各个阶段都得到了很好的界定。生殖细胞是同步发展的囊肿内通过精子沿睾丸的长阶段的进展顺序。在博个有丝分裂和雄性生殖细胞的减数分裂，胞质分裂发生不完全的，使得子细胞仍然被称为环管（ 图1）的细胞质桥相连。睾丸的顶端尖包含生殖系干细胞的细胞群，使人们产生精原细胞，经过4有丝分裂不完全胞质分裂，以产生初级精母细胞的16 - 细胞包囊。经过减数分裂S期，初级精母细胞进入G2，中〜90小时期间，细胞体积增大〜25倍的长期增长期。进展通过减数分裂Ⅰ和减数分裂II结果在次级精母细胞和精子细胞单倍体64细胞囊肿，分别为32个细胞囊肿的形成。不成熟的，圆形精子细胞进行广泛的细胞重塑，形成成熟的精子。减数分裂后的细胞，尤其是拉伸和束成熟的精子细胞，占据大部分睾丸的体积。 Ŧ功能性精子雌蝇他成功的运输要求男性生殖系统，它是由几个配对结构（睾丸，精囊和附腺）和单射精管的（ 图2）的不同部分之间的协调。精子在睾丸内产生和精囊，直到交配24内存储。附件包含腺体产生精液分泌细胞。精子从精囊迁移混有射精管，它同时连接到精囊和附腺内精液。精子和精液的该混合物最终泵送出阳入阴穿过射精管灯泡位于的雄性腹部25的后端飞的阴道。精液中的蛋白质是内中的代表称为精囊特殊器官长时间贮存精子必需的果蝇雌性26 roductive道。 可在科学文献中3-12优良方法果蝇睾丸中分离并在细胞精子发生的不同阶段的可视化。这里，我们通过介绍这些协议的例子，并附带视频演示添加到这个知识体。的协议，用于制备活睾丸样品相衬显微镜是根据先前描述的方法27。该协议为甲醛固定和睾丸的免疫染色也基于先前描述的方法28。本文所描述的方法已被用于在果蝇精子发生的许多研究（例如，以评估动力蛋白的作用，一个负端定向微管运动，在果蝇的精子）。 除了基本的协议中，提供了varyin建议克解剖，以丰富的精原细胞，精母细胞，或成熟的精子。不同的方法，用于处理睾丸，使得囊肿要么保持不变，或者根据需要被描述被打乱。在使用果蝇睾丸作为模型系统的优点在于，相比于果蝇卵母细胞和胚胎中，抗体和染料可以很容易穿透细胞之后从睾丸其扩散，并需要较少的洗涤步骤，因此，协议可以在一个比较来进行很短的时间。

<table border="0" cellpadding="0" cellspacing="0" width="1013">
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			<td height="61" style="height:61px;width:351px;">Sylgard</td>
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			<td style="width:127px;">SYLG184</td>
			<td style="width:321px;">Two-part silicon elastomer for making silicone-coated dissection dish from Kimax Petri dish</td>
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			<td height="21" style="height:21px;width:351px;">PAP pen</td>
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			<td style="width:127px;">NC9888126</td>
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			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI</td>
			<td style="width:215px;">Life Technologies</td>
			<td>P36931</td>
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			<td height="21" style="height:21px;">Mouse anti-gamma-tubulin antibody (clone GTU-88)</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td>T6557</td>
			<td style="width:321px;"></td>
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			<td height="21" style="height:21px;">Cy3-AffiniPure Goat Anti-Mouse IgG&#160;</td>
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			<td height="21" style="height:21px;width:351px;">Triton X-100</td>
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			<td height="21" style="height:21px;width:351px;">16% Formaldehyde</td>
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			<td height="41" style="height:41px;width:351px;">Sigmacote</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:127px;">SL2</td>
			<td style="width:321px;">Use according to manufacturer&#39;s directions to siliconize cover slips</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">DAPI</td>
			<td style="width:215px;">Sigma-Aldrich</td>
			<td style="width:127px;">D-9542</td>
			<td style="width:321px;">0.5 mg/ml in 75% ethanol; store at -20&#176;C</td>
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			<td style="width:127px;">S23020</td>
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			<td height="21" style="height:21px;width:351px;">NaH2PO4</td>
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			<td height="21" style="height:21px;">Single frosted microscope slides</td>
			<td>Corning</td>
			<td>2948-75X25</td>
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			<td style="width:321px;">Optional (to replace untreated microscope slides )</td>
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			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">12-640</td>
			<td style="width:321px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Kimax Petri dish</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">S31473</td>
			<td style="width:321px;">Kimble #23060 10015 EMD</td>
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			<td>Dumont</td>
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			<td>Pattern 5 INOX</td>
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			<td height="21" style="height:21px;width:351px;"></td>
			<td style="width:215px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Name of Equipment</td>
			<td style="width:215px;">Company</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Stemi 2000-CS stereoscope</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
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			<td style="width:215px;">Nikon</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Plan-Fluor 40x objective</td>
			<td style="width:215px;">Nikon</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Axiophot</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
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			<td height="21" style="height:21px;width:351px;">Plan-Neofluar Ph2 40x objective</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></td>
			<td style="width:321px;"></td>
		</tr>
	</tbody>
</table>

cytological analysis of spermatogenesis: live and fixed preparations of drosophila testes

果蝇的是，已被广泛用于澄清各种生物过程的一个强大的模型系统。例如，无论是女性和果蝇雄性生殖系的研究已经极大地促进了减数分裂目前了解以及干细胞生物学。优良协议可用文献中的果蝇卵巢和睾丸3-12的分离和成像。本文中，对于夹层及其制备果蝇睾丸进行显微分析方法，并附带视频演示说明。一种协议，用于从成年男性的腹部隔离睾丸和准备用相差显微镜分析，以及一个协议，用于固定和免疫睾丸通过荧光显微镜分析活组织的幻灯片介绍。这些技术可以在果蝇的突变体，表现出D的特征被应用EFECTS在精子发生，以及在蛋白质的亚细胞定位的可视化。

一个正常解剖对果蝇雄性生殖器官的一个例子示于图2A中 。从成年雄性苍蝇的腹部取出睾丸，通常通过一对精囊（蓝色， 图2A&#39;）连接到射精管（褐色， 图2A&#39;）和一对副腺的（绿色， 图2A&#39;） 。从最所附体组织，射精管和副腺的分离睾丸应分离并弃去，使得只有一对睾丸和精囊腺留（ 图2B和2B&#39;）。精囊?...

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Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

方法分离和制备果蝇睾丸样品（住和固定的），用于成像由相衬和荧光显微术在本文中描述。

精子发生的细胞学分析：Live和固定制剂<em&gt;果蝇</em&gt;睾丸

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

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Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

25.7K 次观看.  Vanderbilt University Medical Center. 方法分离和制备果蝇睾丸样品（住和固定的），用于成像由相衬和荧光显微术在本文中描述。 