Name: cytological analysis of spermatogenesis: live and fixed preparations of drosophila testes
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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?...

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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。

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		<tr height="21">
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			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">NC9888126</td>
			<td style="width:321px;">Ted Pella #22309</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Clear nail protector</td>
			<td style="width:215px;">Wet n Wild</td>
			<td style="width:127px;">7780235001</td>
			<td style="width:321px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">ProLong Gold Antifade Reagent with DAPI</td>
			<td style="width:215px;">Life Technologies</td>
			<td>P36931</td>
			<td style="width:321px;"></td>
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		<tr height="21">
			<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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		<tr height="21">
			<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>
			<td style="width:321px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Triton X-100</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">BP151-100</td>
			<td style="width:321px;"></td>
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			<td height="21" style="height:21px;">Ethanol</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">AC61511-0040</td>
			<td style="width:321px;"></td>
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			<td height="21" style="height:21px;width:351px;">Methanol</td>
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			<td style="width:127px;">A412-4</td>
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		<tr height="21">
			<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>
			<td style="width:321px;"></td>
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		<tr height="41">
			<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;">Na2HPO4</td>
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			<td height="21" style="height:21px;width:351px;">NaH2PO4</td>
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			<td height="41" style="height:41px;width:351px;">BSA</td>
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			<td style="width:127px;">A30075</td>
			<td style="width:321px;">Molecular biology grade</td>
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			<td height="41" style="height:41px;">Glass Coplin staining jar, screw cap</td>
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			<td>70315</td>
			<td style="width:321px;"></td>
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			<td height="21" style="height:21px;">Single frosted microscope slides</td>
			<td>Corning</td>
			<td>2948-75X25</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Poly-L-lysine coated microscope slides</td>
			<td>Polysciences, Inc.</td>
			<td>22247-1</td>
			<td style="width:321px;">Optional (to replace untreated microscope slides )</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Square cover glass</td>
			<td>Corning</td>
			<td style="width:127px;">2865-22</td>
			<td style="width:321px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:351px;">Razor blades</td>
			<td style="width:215px;">Fisher Scientific</td>
			<td style="width:127px;">12-640</td>
			<td style="width:321px;"></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Dumont</td>
			<td>52100-51S</td>
			<td>Pattern 5 INOX</td>
		</tr>
		<tr height="21">
			<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;"></td>
			<td style="width:215px;"></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;">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 height="21" style="height:21px;width:351px;">Eclipse 80i</td>
			<td style="width:215px;">Nikon</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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			<td height="21" style="height:21px;width:351px;">Axiophot</td>
			<td style="width:215px;">Carl Zeiss</td>
			<td style="width:127px;"></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>
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			<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 ...

Research

JoVE Journal

Biology

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

Thin Sectioning of Slice Preparations for Immunohistochemistry

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

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

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

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

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

Isolation of Drosophila melanogaster Testes

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

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

Preparing Individual Drosophila Egg Chambers for Live Imaging

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

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

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

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

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

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

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

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 ...