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. 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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>
			<td style="width:215px;">Jackson ImmunoResearch</td>
			<td>115-165-003</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>
			<td style="width:215px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">28908</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 height="21" style="height:21px;width:351px;">NaH2PO4</td>
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			<td>70315</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>Corning</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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			<td style="width:321px;"></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>
		</tr>
		<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>
		</tr>
		<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?...

Watch this Scientific Journal Video about Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes at JoVE.com

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

25.9K 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。 

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

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

薄切片进行免疫组化切片的筹备工作

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

科研

生物学

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

发展和成人的制备果蝇大脑和视网膜实时成像

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

分离果蝇睾丸

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

裂殖酵母的活细胞定量荧光显微镜分析

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

准备个人果蝇蛋钱伯斯实时成像

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

GFP标记的蛋白质在实时成像<em&gt;果蝇</em&gt;卵母细胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

凋亡细胞清除过程中实时成像<em&gt;果蝇</em&gt;胚胎发育

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

Flow cytometry has been widely used to obtain information about DNA content in a population of cells, to infer relative percentages in different cell cycle phases. This technique has been successfully extended to the mitotic tissues of the model organism Drosophila melanogaster for genetic studies of cell cycle regulation in vivo. When coupled with cell-type specific fluorescent protein expression and genetic manipulations, one can obtain detailed information about effects on cell number, cell size and cell cycle phasing in vivo. However this live-cell method has relied on the use of the cell permeable Hoechst 33342 DNA-intercalating dye, limiting users to flow cytometers equipped with a UV laser. We have modified this protocol to use a newer live-cell DNA dye, Vybrant DyeCycle Violet, compatible with the more common violet 405nm laser. The protocol presented here allows for efficient cell cycle analysis coupled with cell type, relative cell size and cell number information, in a variety of Drosophila tissues. This protocol extends the useful cell cycle analysis technique for live Drosophila tissues to a small benchtop analyzer, the Attune Acoustic Focusing Cytometer, which can be run and maintained on a single-lab scale.

Live Cell Cycle Analysis of Drosophila Tissues using the Attune Acoustic Focusing Cytometer and Vybrant DyeCycle Violet DNA Stain

A protocol for cell cycle analysis of live Drosophila tissues using the Attune Acoustic Focusing Cytometer is described. This protocol simultaneously provides information about relative cell size, cell number, DNA content and cell type via lineage tracing or tissue specific expression of fluorescent proteins in vivo.

活细胞周期分析<em&gt;果蝇</em&gt;组织使用同调声聚焦流式细胞仪Vybrant的DyeCycle紫DNA染色

Flow cytometry has  been widely used to obtain information about DNA content in a population of  cells, to infer relative percentages in different cell ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...