Name: immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes
Uploaded: 2020-08-27T14:00:00
Description: Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

我们感谢P.韦里泽，J.约翰森H.，奥库拉的抗体和A.德贝克，布卢明顿和维也纳的苍蝇库存中心。感谢杰克逊的建议和刺激的讨论。

1. 德罗索菲拉睾丸收藏
注：年轻男性（0-15小时后关闭）是检查双体生殖细胞（即精子卵细胞和精子细胞）所必需的。
<ol><li>尽可能多地选择幼蝇，以尽量减少来自发育精子的干扰。</li>
	<li>麻醉苍蝇通过短暂接触二氧化碳和转移到飞垫14，15。</li>
	<li>在解剖显微镜（10倍放大），使用钳子去除头部。</li>
	<li>将 5 到 10 只被斩首的?...

<ol>
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(51), e2641 (2011).</li><li>Gigliotti, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nup154,+a+new+Drosophila+gene+essential+for+male+and+female+gametogenesis+is+related+to+the+nup155+vertebrate+nucleoporin+gene.">Nup154, a new Drosophila gene essential for male and female gametogenesis is related to the nup155 vertebrate nucleoporin gene.</a> Journal of Cell Biology. 142 (5), 1195-1207 (1998).</li><li>Chen, H., Chen, X., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+lamina+regulates+germline+stem+cell+niche+organization+via+modulation+of+EGFR+signaling.">The nuclear lamina regulates germline stem cell niche organization via modulation of EGFR signaling.</a> Cell Stem Cell. 13 (1), 73-86 (2013).</li><li>Fawcett, D. W., Chemes, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+distribution+of+nuclear+pores+during+differentiation+of+the+male+germ+cells.">Changes in distribution of nuclear pores during differentiation of the male germ cells.</a> Tissue Cell. 11 (1), 147-162 (1979).</li><li>Bolte, S., Cordelieres, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guided+tour+into+subcellular+colocalization+analysis+in+light+microscopy.">A guided tour into subcellular colocalization analysis in light microscopy.</a> Journal of Microscopy. 224, 213-232 (2006).</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 (1), 11-22 (2012).</li><li>Bonaccorsi, S., Giansanti, M. G., Cenci, G., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Immunostaining of Drosophila testes. 2011 (10), 1273-1275 (2011).</li><li>Bonaccorsi, S., Giansanti, M. G., Cenci, G., Gatti, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Paraformaldehyde fixation of Drosophila testes. 2012 (1), 102-104 (2012).</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=Spermatogenesis:+analysis+of+meiosis+and+morphogenesis.">Spermatogenesis: analysis of meiosis and morphogenesis.</a> Methods in Molecular Biology. 247, 45-75 (2004).</li><li>Kibanov, M. V., Kotov, A. A., Olenina, L. 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该协议为成功免疫全安装成人嗜血杆菌睾丸提供了一种方法。正如在其他程序中报告，年轻男性必须使用（我们甚至缩短年龄到20小时后，关闭），以尽量减少发展精子的存在，这有可能阻碍精子细胞。解剖应快速进行，抗体稀释必须进行调整，以优化信号与噪声比21、22、23。因为使用其他协议，我们没有观察到整个睾丸深度?...

果蝇睾丸是研究核建筑的宝贵模型。在线粒体精子癌细胞中，核体积主要被色质所占据。这些细胞经过四轮分裂，产生16个原发性精子细胞的囊肿。在接下来的几天里，精子细胞通过6个发育阶段（S1-S6），体积大大增加，大约是20倍1，2。它们也改变了其核形态。层压 Dm0 揭示的原子核轮廓变得不规则，并显示深层入侵1，3。伴随着广泛的基因表达变化和改变染色质组织1，4，5，6，7。相间染色体在S4-S5阶段定义明确，形成三个不同的质量，对应于2个主要双价自体体和与核对接的X-Y对。
Mad1 最初被隔离为线粒体检查点的关键部分（在8中审查）。在相间，它被描述为主要位于核信封，但也在核质9，10，11。在嗜血杆菌睾丸中，我们报告说，Mad1在核质中很突出，与两种主要的自体染色质质量密切相关，在较小程度上与XY色素有关。我们定义这种染色质相关结构为MINT（含Mad1核内领土）12。其他四种蛋白质与精子细胞中的MINT相关：核苷Mtor/Tpr、SUMO肽酶乌尔普1、线粒体检查点蛋白Mad2和类似聚团的复合Raf212的亚单位。
为了完成对MINT的描述，我们需要使用抗体，并面临着睾丸中免疫稳定通常遇到的技术问题：渗透性差，导致睾丸深度的污渍严重不均匀，特别是在 大型精子细胞中。S消解制剂 增加了抗体访问，但 导致压缩图像，限制3D分析， 并 防止定量荧光的测量 ，包括同位素化。抗体渗透的问题也可以通过渗透剂，如0.3%纳脱氧色素13来解决，但这个程序并没有在我们手中产生可重复的结果。因为我们进行了许多联合标记实验，我们期待改进渗透协议。1% NP40 加希普坦的组合导致更多的可重复性，特别是在研究大型精子细胞方面。
本协议对悬浮睾丸进行固定和免疫维护，提高样品质量，提高可重复性，使其适合共聚焦显微镜。我们利用该协议更好地定义某些核包络蛋白与大型精子细胞核质结构的空间关系。这种方法将允许更好地研究伴随精子细胞发育的变化，例如细胞核的动态结构变化，包括色素、核质和核毛孔。

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Drosophila srains</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ketelGFP y; ketelGFP/y+CyO</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>Gift from A. Debec (Institut Jacques Monod, UMR 7592, Paris, France)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Mad1-GFP</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>J Cell Sci. 2011 May 15;124(Pt 10):1664-71.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Mtor RNAi&#160;</td>
			<td style="width:129px;">VDRC</td>
			<td style="width:93px;">ID 110218</td>
			<td>Vienna Drosophila RNAi Center</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Materials</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:277px;">DAPI&#160;</td>
			<td style="width:129px;">Thermo</td>
			<td>D1306</td>
			<td>Stock solution must be prepared in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dumont # 5 Fine Forseps&#160;</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MBP Wide Bore Pipette Tips</td>
			<td style="width:129px;">Fisherbrand</td>
			<td>11917744</td>
			<td>Wide aperture tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#160;0.2 mL Individual Tube</td>
			<td style="width:129px;">Thermo</td>
			<td>AB-0620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml micro centrifuge tubes&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td>T3531-200EA&#160;</td>
			<td>Siliconized 2ml tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citifluor AF1</td>
			<td style="width:129px;">Citifluor</td>
			<td>AF1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dakopen&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:93px;">Z377821-1EA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum (NGS)</td>
			<td style="width:129px;">Sigma</td>
			<td>NS02L-1ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 4%</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:93px;">P6148-500G&#160;</td>
			<td>Paraformaldehyde (4%) dissolved in PBS 1X&#160;; aliquot by 600 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1X</td>
			<td style="width:129px;">Thermo</td>
			<td>14190094</td>
			<td>DPBS, no calcium, no magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% (0.2 or 0.3%) PBS-T&#160;</td>
			<td style="width:129px;"></td>
			<td></td>
			<td>PBS 1X&#160; containing 0.1% (0.2 % or 0.3%) Triton X-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100, for molecular biology,&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP booster</td>
			<td style="width:129px;">Chromotek</td>
			<td>gba488</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Mtor</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;">1/40 gift from J. Johansen, Iowa State University</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti-Nup62</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;">1/200 gift from H. Ohkura (University of Edinburgh, UK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guinea-pig anti-Ulp1</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Rabbit anti-Raf2</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DyLight conjugated series&#160;</td>
			<td style="width:129px;">Thermo</td>
			<td style="width:93px;"></td>
			<td>1/500 Donkey anti-(guinea-pig, mouse,&#160; rabbit or rat) as second antibodies</td>
		</tr>
	</tbody>
</table>

immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes

嗜血 性睾丸是研究生物过程的强大模型系统，包括干细胞生物学、核结构、肌瘤和精子发育。然而，由于抗体渗透，整个 嗜血杆菌 睾丸的免疫带通常与染色的严重不均匀性有关。压扁的准备工作只是部分克服了这个问题，因为它降低了分析的3D质量。在此，我们描述了使用NP40和六烷在固定过程中与液体介质免疫带的全安装协议。它保留了适合共焦显微镜的体积以及可重复和可靠的标签。我们展示了不同的例子，从共聚焦部分的精子细胞核的3D重组。睾丸内部和睾丸间可重复性允许不同基因型的单个细胞之间的荧 光 进行3D定量和比较。我们使用了核内造币厂结构（含Mad1的核领土内部）的不同组件以及与核孔复合物相关的两个组件来说明该协议及其在睾丸最大细胞S4-S5精子细胞上的应用。

获得充分染色的Drosophila睾丸的主要障碍是抗体的渗透性有限，通过使用压扁制剂部分解决，但代价是导致对3D分析的限制（例如3D表示或同地测量）。该程序允许统一标记，并保留睾丸所有细胞的体积。在这里，我们重点介绍了S4-S5精子细胞的3D表示方法，因为它们是最大的细胞，因此对挤压变形更敏感。我们使用了不同的核标记，包括核苷酸和核内薄荷域（含Mad1核领土）的标签，由线粒体蛋白Mad...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

我们在这里提出了一个改进的协议，为全安装准备和免疫染色的 嗜血杆菌 睾丸，适合共焦显微镜，也允许可重复和可靠的标签。我们通过对大型S4-S5精子细胞的同位素化实验进行3D表示和量化来说明此协议。

用于大型精子细胞三维共焦分析的全山 嗜血杆菌 睾丸免疫染色

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

Research

JoVE Journal

Biology

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Double Whole Mount in situ Hybridization of Early Chick Embryos

双整装原位杂交技术在早期鸡胚

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Method for Whole Mount Antibody Staining in Chick

整装在鸡的抗体染色方法

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

在整个猴脑进行大规模蛋白质检测的批次免疫染色

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

全安装胚胎肢体皮肤血管的免疫组织化学分析：一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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 ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

全山原位杂交E8.5，E11.5小鼠胚胎

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

全山RNA荧光<em在原位</em&gt;杂交<em&gt;果蝇</em&gt;胚胎

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

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

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

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 ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

平山准备观察斑马鱼胚胎标本染色，并通过分析全山<em&gt;原位</em&gt;杂交

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

用于大型精子细胞三维共焦分析的全山 嗜血杆菌 睾丸免疫染色

用于大型精子细胞三维共焦分析的全山嗜血杆菌睾丸免疫染色