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 只被斩首的苍蝇转移到 100 μL 的林格缓冲区（183 mM KCI、47 mM NaCl、10 mM Tris-HCI、1 mM EDTA、完整的蛋白质抑制剂、pH 6.8）在黑色背景支撑下的硅胶涂层玻璃滑梯上。</li>
	<li>解剖显微镜的放大倍数为40倍，使用一对5号钳子在胸腔和腹部之间保持苍蝇，并用其他钳子将腹部从胸腔拉开。迅速将睾丸和相邻组织从飞尸体15中分离出来，并在同一滑梯上放置一滴新的林格缓冲器。</li>
	<li>所有睾丸准备好后，从剩余的组织（辅助腺体和外生殖器）中清洁睾丸。</li>
	<li>从包含解剖睾丸的滴中取出多余的缓冲区。通常，使用 p200 移液器，在 200μL 尖端顶部安装 10 μL 尖端。这允许通过一个小孔径去除多余的液体。将4%的甲醛固定在含有解剖睾丸的液滴上，然后迅速将睾丸转移到含有新鲜固定溶液的管子上。</li>
</ol>2. 甲醛固定
<ol><li>使用前准备 2 mL 的固定溶液:在磷酸盐缓冲盐水 （PBS） 中稀释 4% 副甲醛的 600 μL，其中 30 μL 的 NP40 20% 加 1200 μL 的乙烷在 2 mL 硅化微中微富格管中。在 600μL 单环中使用 4% 的 PBS 中副甲醛，储存在 -20°C。 注意:甲烷和甲醛是有毒的，应在烟灰罩中处理。按照主办机构的规定处理。</li>
	<li>使用带宽孔径尖的 p200 微管将睾丸转移到 PFA/NP40/heptane 解决方案。用手上下摇动管子30s。在室温下继续将睾丸固定在旋转搅拌机上 30 分钟。</li>
	<li>停止旋转搅拌机，将管子放在管架中，以便相分离，并让睾丸沉淀到管的底部。</li>
	<li>取出所有六烷和固定溶液，用 1 倍 PBS 快速冲洗睾丸三次。</li>
	<li>转移到一个干净的200μL单管。在这些小管中执行所有孵化，以最大限度地减少所使用的抗体量。</li>
	<li>使用附带 P200 和 P10 提示的 200μL 微皮带去除多余的缓冲区。小心不要吸气睾丸。</li>
	<li>将睾丸放在冰上1x PBS中，直到所有样品都准备好。</li>
</ol>3. 溶液中的抗体染色
<ol><li>丢弃 PBS，在室温下将睾丸留在 0.3% PBS-T 中 30 分钟，以渗透细胞膜。</li>
	<li>在同一缓冲区预孵化，加上5%的正常山羊血清（NGS）1小时。</li>
	<li>在 0.3% PBS-T 加上 5% NGS （材料表） 中稀释的主要抗体中加入 200 μL。</li>
	<li>在 4 °C（或室温）下在夜间孵化主要抗体，在摇杆上轻轻搅拌。</li>
	<li>在室温下在摇动器上用 0.3% PBS-T 清洗 3 次 15 分钟。允许睾丸按重力沉降到管子底部。</li>
	<li>从 DyLight 组合系列中添加 200 μL 的二次抗体稀释 1:500。</li>
	<li>在室温下在暗室中孵育二次抗体4小时。</li>
	<li>在室温下在摇动器上清洗 0.3% PBS-T 15 分钟。然后重复0.2%PBS-T和0.1%PBS-T，每次在室温下在摇动器上30分钟。在 1 倍 PBS 中进行 30 分钟的最终洗涤，以去除洗涤剂。</li>
	<li>在 1 倍 PBS 中以 1μg/mL 的速度加入 180μL 的 DAPI 解决方案，15 分钟。库存溶液为 H 2 O 中的10毫克/mL，避免在 PBS 中制作库存溶液，从而减少较大精子细胞中扩散染色质上的 DAPI 染色信号。</li>
	<li>每次洗3次5分钟。</li>
	<li>使用疏水屏障笔在干净的滑梯上画一个圆圈。</li>
	<li>吸气睾丸，并把它们放在幻灯片上。用 0.1% PBS-T 预冲洗宽孔径尖可防止睾丸粘在尖端上。删除多余的 PBS。</li>
	<li>添加一滴（约100μL）的花旗流体AF1。</li>
	<li>轻轻将玻璃盖在纸巾上，小心避免诱捕气泡。</li>
	<li>使用透明指甲油将盖片密封在滑梯上。</li>
	<li>观察显微镜上的幻灯片。</li>
</ol>4. 同地分析
<ol><li>获取对焦图像。我们使用了蔡司 LSM 710 共焦显微镜 （63x 计划杏色油）， z 分辨率为 0.5 或 1 μm。</li>
	<li>从独立睾丸中选择不同的细胞。</li>
	<li>将图像导入到处理软件（例如，Imaris）。</li>
	<li>通过手动分割来定义原子核，以排除相邻的核。使用科洛克软件;它收集皮尔逊的相关系数（PCCs）在整个体积之间的2个氟磷。</li>
</ol>

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作者没有什么可透露的。

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的污渍，我们开发了一种固定方法，将PFA与NP40和希普坦结合在一起。我们现在在PBS中通常使用600微升的4%甲醛，30微升为20%NP40，外加两卷乙烷。这对于允许足够的渗透至关重要。将 NP40 的量降低到 10 μL 明显减少了标签。所有后续步骤均在悬架中进行，通过允许睾丸通过重力轻轻地下降到管子底部来消除多余的缓冲。对于将样品转移到幻灯片的最后一步，我们将在 0.1% PBS-T 中冲洗尖端，以防止睾丸粘在圆锥体上。根据观察，免疫遗传学染色协议与各种核蛋白配合得很好，包括此处使用的标记物，以及表观遗传性高石修饰、层压素和核蛋白的探针。我们注意到，该协议也可用于幼虫睾丸，在这种情况下，应省略六烷，因为它使睾丸更难恢复完好无损。
该协议特别旨在保持未损坏的精子细胞堆栈在一起，以高效和统一的睾丸标签。我们用大型晚期精子细胞来说明协议，其畸形细胞核使它们成为最难保存的细胞之一。此外，在这些精子细胞中，核标签往往变得稀释，可能是因为核的大小。例如，人们通常观察到，DAPI或层压层压标签在睾丸顶点的细胞中更强，随着精子细胞的生长，它们会逐渐变弱和分散。这也可能是核蛋白的分布只在早期细胞（精氨酸和早期精子细胞）17中描述的原因之一。
已发布Drosoophila S5精子细胞免疫染色的优秀图像，特别是针对睾丸特异性t-BRD和丹尼蛋白25，26使用0.3%脱氧酸钠协议或在组织21，22挤压后。在我们手中，挤压会导致器官深处染色的严重不均匀性，并牺牲 3D 结构。即使使用0.3%的脱氧酸钠能产生更好的效果，我们仍然观察到不均匀性。因此，我们更喜欢NP40的可重复性和标签质量。共焦图像允许重建核蛋白质的3D图像。例如，我们成功地应用了这种方法使用许多不同的探针，这里通过核内薄荷域，与自体色质和核孔网络紧密相关。这些精子细胞核核毛孔的3D分布令人着迷，现已可供进一步研究。图像允许从来自同一睾丸和不同睾丸的不同精子细胞中重复量化同位素。据我们所知，这是首次对此类样本测量皮尔逊相关系数的准确量化。它提供了准确对不同睾丸进行免疫蛋白水平比较所需的信心。

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。

<table border="0" cellpadding="0" cellspacing="0" style="width:1005px;" width="1005">
	<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Mad112描述。由于 MINT 可以通过几个不同的抗体揭示出来，我们使用它们来测试定量 3D 共定位协议的坚固性，这种方法需要正确的空间分辨率。
上述协议允许获取质量足以进行 3D 重组的共焦图像。舞台 S5 精子细胞的四个相邻序列部分在 图 1A中描述，显示 MINT（使用红色 GFP 助推器进行 Mad1 免疫增压）与 DAPI（青色）的代表性免疫染色。染色体被组织成特征的3个主要不同的簇，MINT结构在DNA周围以精细的细节显示，与自体染色质质量密切相关，但与自身色素质量不同。该决议还允许在核外围以及2号和3号染色体的自动体质量之间检测Mad1的存在。我们使用伊马里斯软件进行3D表示。核表面由 Mad1 在核信封（灰色）的低水平存在定义，MINT 的定义是核心（红色）内部的 Mad1 与 DAPI 染色（蓝色）（图 1A）的高度积累。3D 重组电影很好地说明了 Mad1（在核信封和 MINT）与 S4–S5 精子细胞阶段染色体的体积和关系。
核素的分布只是这些细胞中描述得不好，可能是由于在这些大畸形细胞核中检测它们的困难。Nup154的分布只报告过一次以前16和Nup153和Nup98只研究在精子和小，早期精子细胞17。由核苷Nup62和进口β（ketel基因的产物）染色的大型S5精子细胞的3D重组是可行的，分别表现在图1C和图1D中。这些核孔相关部件似乎不均匀地分布在核边缘，作为不规则大小的点。例如，进口β聚集在自体色素周围，类似于已经描述的层压网3。这种分布与孔隙丰富的地区和大孔无孔区域已经描述电子显微镜在啮齿动物精子细胞18。了解层压素和核蛋白分布的变化将有助于了解这些G2细胞在男性髓病之前的核心包络结构。
在保存细胞的3D体积的同时，该协议还允许抗体均匀地渗透到每个细胞的深度，从而允许定量分析，如同位素化。皮尔逊相关系数 （PCC） 是一个成熟的指标。它可以从 +1 （表示完美的正相关） 到−1 （完美的负相关）19。为了评估与Mad1相比，MINT伙伴蛋白在精子细胞中的本地化程度，我们之前在S5精子细胞12（图2A，B）中使用图像J在0.5μm的单节上估计了PCC。我们通过在整个核上而不是仅仅在表面上使用 3D 共定位来更深入地测试协议。核的体积由手动分割定义，PCC 使用选址工具进行测量。我们分析了8个S5精子细胞核，来自相同的睾丸或不同的睾丸。图 2B显示了一个核心的 3D 表示，该原子核有助于测量 PCC。图2D显示PCC从三个不同的睾丸中对8个不同的S5精子细胞核的测量。PCC 值没有不同，从 0.6 和 0.75 不等，表示共同定位的高价值。这些与单个部分定义的 PCC 没有区别。因此，该程序允许重复的均匀标签，并保留了睾丸所有细胞的良好体积。
由于该协议允许从独立测试中可靠测量同位素化，因此允许比较不同苍蝇基因型的染色。这里通过比较WT苍蝇和RNAi耗尽Mtor（由dsRNA UAS线和Bam-Gal4驱动程序的表达产生，在晚精子古氨和早期精子细胞20中实现大量耗竭）来说明这一点。正如同位素化实验所述，Mtor-RNAi耗竭的睾丸和野生类型睾丸被解剖和固定在一起，并在恒定的设置下获取共焦图像。尊重这些条件，个别细胞的比较成为可能。我们观察到，MINT比核信封受到Mtor损失的影响更大，如具有代表性的Mtor RNAi耗竭（上）和wt（下）细胞（图3）12所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61061/61061fig01.jpg"> 图1:使用不同探头对 嗜血杆菌 S4-S5精子细胞核进行免疫染色图解。（A） 单阶段 5 精子细胞核免疫的图像，与α-GFP 抗体 （GFP 助推器） 一起检测由 Mad1-GFP （红色） 到 DAPI 染色 （蓝色） 的 MINT 结构。它准确地显示了Mad1和两个自体染色质群（作为星号）之间的关系。在核的外围也隐约检测到 Mad1。所有图像都是连续 3 （0.5 μm） 连续共焦切片的 Z 投影。共聚焦图像是在共焦显微镜上获得的（63倍，计划杏色油DIC目标透镜）。比例杆是5μm.（B） 帧从电影渲染相同的精子细胞核。核表面由核信封上的 Mad1 低表达（灰色）定义，MINT 由核内的 Mad1 高表达（红色）定义。（A， B）转载，并获得先前公布的数字（从12）的许可。（C） 第 5 阶段精子细胞核的 3D 图像，同时免疫维持 Nup62（黄色）和 MINT（红色 GFP 助推器）。比例尺是4μm.（D） 第5阶段精子细胞核的3D图像，同时免疫维持进口β Ketel（黄色）与DNA（蓝色DAPI）。比例栏为 5μm。 <a href="https://www.jove.com/files/ftp_upload/61061/61061fig01large.jpg" target="_blank">请单击此处查看此图的更大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61061/61061fig02.jpg"> 图2:2D和3D共定位量化的Mad1与乌尔普1。（A） 与 GFP 助推器抗体检测 Mad1 （绿色） 和抗 Ulp1 抗体 （红色） 代表的 1μm 厚的精子细胞核的阶段 5 精子细胞核的合并图像。右侧表示使用 Coloc-2 图像J 插件计算的皮尔逊强度相关系数。PCC估计为0.73（经Raich12许可转载）。（B） 与GFP助推器抗体检测Mad1（绿色）和以3D投影中表示的抗Ulp1抗体（红色）的5级精子细胞核（与相邻表中的G核相对应）的合并图像进行免疫。原子核的体积由手动分割定义。在右边，表列出了Pearson强度相关系数，这些系数计算在8个不同的精子细胞（A到H）上，从3个不同的睾丸（1到3）。 <a href="https://www.jove.com/files/ftp_upload/61061/61061fig02large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61061/61061fig03.jpg"> 图3:与野生类型相比，对经Mtor RNAi治疗的精子细胞进行免疫荧光分析。使用 MtorRNAi （ID 110218） 细胞 （上面板） 和野生类型细胞 （下面板） 耗尽的 Mtor 细胞被染色为 Mad1、 Raf2 和 Mtor 蛋白质。睾丸被固定和染色在一起，安装在同一滑梯上，图像获得相同的参数。RNAi细胞可以分别因Mad1-GFP的缺失或存在而与WT区分开来。该数字代表最大预测 4 x 0.5 μm 堆栈。比例尺为 5 μm（在获得先前公布的12个数字的许可后转载）。 <a href="https://www.jove.com/files/ftp_upload/61061/61061fig03large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

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 ...

immunostaining-whole-mount-drosophila-testes-for-3d-confocal-analysis

Universidad San Sebastian

Research

JoVE Journal

Biology

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

4.1K 次观看.  Université de Paris, F 75006, Paris, France. 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。

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

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

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

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

整装在鸡的抗体染色方法

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

斑马鱼整装高分辨率双荧光原位杂交

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

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 RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

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

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

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

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

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 in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

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

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

定量，单细胞在全安装胚珠在染色质修饰和核体系结构分析的有效方法<em&gt;拟南芥</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

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

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

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

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

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 ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

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