作者感谢所有同意并为这项研究捐赠样本的女性。

研究内容和方案已通过深圳市中山泌尿外科医院研究伦理委员会的伦理审查和批准。参与研究的所有女性（20-40 岁）都对样本收集和使用提供了知情同意。
1.病理组织的获取
<ol><li>准备用于组织采集的工具，即测量尺、镊子、包埋盒、包埋纸和纸巾篮。</li>
	<li>观察使用移液管的标准方法收集的子宫内膜组织（比绿豆大）的数量是否足够。</li>
	<li>用镊子将福尔马林的子宫内膜组织转移到包埋纸上，并用尺子测量子宫内膜组织尺寸。</li>
	<li>用包埋纸包裹子宫内膜组织，然后放入包埋盒中。</li>
	<li>将包埋盒放入纸巾篮中进行脱水。</li>
</ol>2.组织脱水
<ol><li>将组织篮放入脱水机的反应室中（参见 材料表），并开始常规组织脱水程序：福尔马林100分钟;福尔马林100分钟;75%酒精60分钟;85% 酒精 60 分钟;95% 酒精 60 分钟;100%酒精60分钟;100%酒精60分钟;100%酒精60分钟;二甲苯35分钟;二甲苯20分钟;二甲苯20分钟;蜡80分钟;蜡80分钟;打蜡 80 分钟。该过程大约需要15小时。</li>
	<li>在组织脱水程序结束时，打开脱水机的反应室，取出组织篮。</li>
</ol>3. 组织包埋
<ol><li>根据试样尺寸取出合适的包埋模具，在70°C下填充石蜡。</li>
	<li>快速将纸巾放入模具中并仔细调整，使纸巾位于模具的中心。</li>
	<li>将模具平稳地移动到冷却板上，轻轻按压纸巾，同时底部的石蜡凝固。</li>
	<li>将包埋盒放在模具顶部，并加满更多的蜡。</li>
	<li>将模具放在冷却板上，当石蜡完全凝固时，将带有连接盒的块从模具上取下。</li>
</ol>4. 组织切片
<ol><li>将块插入切片机的样品夹上，将刀片放入支架中，调整块平面与刀片之间的角度，将切片厚度调整为4μm，转动手轮，开始切片。</li>
	<li>通过从块上切下几片薄片来暴露适当的组织表面。用刷子取出连续和完整的部分。</li>
	<li>当切出足够多的断面时，停止转动手轮，用镊子去除前端不合格的断面，用镊子和刷子将切片漂浮在水浴中的42°C水面上。</li>
	<li>将部分完全压平后，在防分离载玻片上挑选部分，并转移到65°C的载玻片加热器中烘烤60分钟。</li>
	<li>烘烤完成后，将载玻片从载玻片加热器中取出。</li>
</ol>5.免疫组化染色
<ol><li>用抗体稀释剂将一抗稀释到工作溶液中。详见 表1 。</li>
	<li>将稀释后的抗体溶液放入专用试剂瓶中，将检测试剂盒放入全自动IHC染色仪的试剂室中（见 材料表）。</li>
	<li>将载玻片放在载玻片支架上，用特殊的盖子覆盖，然后将它们插入仪器的实验反应室中。</li>
	<li>仪器自动识别载玻片上的试剂和实验信息后，点击开始按钮 开始 免疫组化染色。实验持续约3小时。</li>
</ol>6.载玻片的脱水和密封
<ol><li>免疫组化染色后，取出载玻片支架，取下专用盖板，将染色的载玻片放入载玻片支架中，用清水洗去载玻片上残留的染料。</li>
	<li>将载玻片架转移到自动盖玻片机上（参见 材料表），选择并运行脱水和密封程序。</li>
	<li>脱水密封后取出载玻片。</li>
</ol>7. 扫描载玻片
<ol><li>将载玻片放在全景病理图像扫描仪的载玻片架上（见 材料表），放入仪器载玻片扫描舱进行全景病理图像扫描。扫描需要 2 分钟。请参阅 图 1。</li>
</ol>8. 图像分析
<ol><li>导入图像<ol><li>打开病理图像分析软件（参见 材料表）并创建一个新文件夹。导入要分析的免疫组织化学图像。</li>
	</ol></li>
	<li>构建组织分类器<ol><li>标记多个组织和空白注释，以训练和建立分类器，分别用于识别组织和空白区域。</li>
		<li>使用实时调优，在标记标注过程中实时观察软件的识别能力。如果识别不及时，则备注注释并再次训练，直到组织识别准确。</li>
	</ol></li>
	<li>构建分析算法<ol><li>根据实验类型在软件中选择标准算法：多重 IHC。</li>
		<li>通过选择典型的负像素和正像素来设置单元格识别的颜色参数。 在此基础上，设置细胞核、细胞质、细胞膜的参数，实时观察细胞识别情况，直到找到最适合图像的参数。</li>
		<li>设置阳性细胞识别阈值，实时观察识别情况，直到调整到合适的阈值。</li>
		<li>在算法中选择组织分类器，在组织分类器中检查组织部分，从而在组织的基础上识别细胞。至此，分析算法的建立就完成了。</li>
	</ol></li>
	<li>运行图像分析<ol><li>选择分析区域。使用已建立的算法分析图像。</li>
	</ol></li>
	<li>软件分析完成后，手动检查图像识别是否准确，包括组织识别、阴性和阳性细胞识别。</li>
	<li>如果图像识别不准确，请重新调整算法和参数阈值，重新运行图像分析，直到成功。导出分析结果。请参阅 图 1。</li>
	<li>其他免疫细胞也可以按照上述步骤进行分析（见图1）。根据不同子宫内膜免疫标志物（CD56+uNK细胞、Foxp3+Tregs、CD68+巨噬细胞、CD163+M2巨噬细胞、CD1a+DCs和CD8+T细胞25,26）的实际表达设置不同的分析参数。</li>
	<li>通过软件的计算，得到子宫内膜免疫细胞的比例（见 表2）。用它来评估复发性流产患者子宫内膜中各种免疫细胞的水平。</li>
</ol>

免疫组织化学染色的子宫内膜组织的图像分析

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作者没有什么可透露的。

该协议建立了数字免疫组化图像分析平台，以定量分析RM患者的子宫内膜免疫细胞。在这里，检测了六种子宫内膜免疫标志物，以评估RM患者的子宫内膜免疫微环境。
黄体中期的接受性子宫内膜是成功植入和怀孕的关键27,28。因此，评估子宫内膜免疫细胞百分比在估计子宫内膜容受性方面起着重要作用。通过传统的病理学方法进行子宫内膜免疫细胞分析是预测性的，因此对临床应用没有帮助。目前，没有标准化的方法来测量免疫细胞百分比，这为理解这些细胞在怀孕中的作用构成了障碍。传统的 IHC 分析基于选定的视野和手动计数。常规 IHC 无法评估免疫细胞的精确组织分割和定位，因为手动分析是主观的，导致观察者偏倚29。
与分析所选领域中免疫细胞的分布相比，组织全景分析对于分析子宫内膜免疫细胞的分布更准确。利用基于机器学习算法的数字病理学方法已在多个肿瘤中进行了测试，以评估大组织区域和复杂的细胞表型30,31。在本研究中，介绍了一种用于获取子宫内膜样本全景图像的商业系统。随后使用基于免疫组化图像分析系统的自动定量分析确定子宫内膜免疫细胞的百分比。
这里使用的免疫组化图像分析系统是专门针对病理组织的图像分析平台，它利用人工智能实现组织分割。已经报道了许多使用该系统的各种模块的研究32,33,34。该系统用于量化使用传统图像处理软件难以分析的各种组织病理学变化和发现。例如，在黄体中期，CD56+NK细胞的数量约占子宫内膜总细胞数的5%。传统的免疫组化分析难以准确计算CD56+ NK细胞的数量，计算整个切片的阳性细胞总数可能需要至少30分钟，但使用这里使用的系统需要2-3分钟。因此，使用这里使用的免疫组化图像分析系统，可以很容易地、准确地分析子宫内膜免疫细胞。通过图像分析对病理结果进行量化，有助于提高子宫内膜免疫环境评估的客观性、精确性和说服力。
然而，所描述的方法存在局限性。子宫内膜由与妊娠结局相关的各种免疫细胞组成。因此，仅定义一种或两种免疫标志物可能是不够的。因此，需要多参数方法来全面评估细胞的免疫谱。
综上所述，可以得出结论，该协议已成功将数字图像分析应用于RM患者黄体中期的子宫内膜切片中，用于免疫细胞的定量，并在本研究中进行了全景分析，以确定子宫内膜CD56 + uNKs，Foxp + Tregs，CD68 +巨噬细胞，CD163 + M2巨噬细胞，CD1a + iDCs和CD8 +的分布黄体中期的T细胞。需要更多的证据来支持子宫内膜免疫细胞与RM患者妊娠结局之间的关联，未来的研究将重点关注这一点。这是第一项使用数字病理学调查RM患者子宫内膜免疫环境的研究。

复发性流产（RM）是指连续两次或两次以上妊娠的流产，是一种复杂的疾病，近年来引起了临床医生的关注。育龄妇女RM的发病率为1%-5%1。既往研究结果表明，免疫因子与RM 2,3,4,5的发病机制密切相关。维持母胎界面的免疫稳态是胚胎植入和发育所必需的。子宫内膜免疫细胞在维持这种稳态方面发挥多种调节作用，例如促进滋养层侵袭、重塑螺旋动脉和促进胎盘发育 6,7,8,9。
RM 女性的子宫内膜免疫细胞异常既往已有报道。结果显示，高密度子宫自然杀伤细胞 （uNK） 与 RM10、11、12 的发生密切相关。据报道，与活产女性相比，RM女性子宫内膜中的巨噬细胞数量有所增加13。调节性T细胞（Treg）在母体对胚胎的免疫耐受中发挥作用，其水平和功能在RM患者的蜕膜中降低14。细胞毒性 T 细胞 （CTL） 和树突状细胞 （DC） 也在妊娠的免疫调节中发挥作用15,16。因此，对黄体中期局部子宫内膜免疫细胞进行全面的定量分析有助于更好地了解RM的发病机制。目前一些用于子宫内膜免疫细胞定量分析的方法使用流式细胞术，它可以准确地标记具有多种标记物的免疫细胞17,18。然而，流式细胞术的临床应用受到限制，因为它只能在新鲜组织上进行。只有当存在大量多余肿瘤时，获得新鲜组织才可行，这在子宫内膜中很少见。免疫组化可以很好地原位观察组织形态，也可以标记各种免疫细胞，而传统的免疫组化技术无法对免疫细胞进行定量分析。
与常规免疫组化实验相比，子宫内膜免疫细胞的定量免疫组化分析具有重要的临床意义。IHC强度评分通常采用四分制或病理诊断和研究中的强弱排序19,20,21。然而，这种半定量技术是主观的、高度不准确的，并显示出显著的观察者内部和观察者间变异性22。一种可能的解决方案是机器学习的应用，这在数字图像分析中很有价值23,24。通过提供定量测量，这种方法可以更精确地评估子宫组织内的免疫细胞浸润、分布和密度。这种定量信息可以帮助阐明月经周期和各种病理条件下免疫细胞群的动态变化。总体而言，通过免疫组化定量分析子宫内膜中免疫细胞的能力为了解子宫的免疫微环境提供了有价值的见解。
因此，该方案旨在开发和验证一种数字免疫组化图像分析平台，以定量分析RM患者黄体中期的子宫内膜免疫细胞，包括uNK细胞、Tregs、巨噬细胞、DC和细胞毒性T细胞。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Automated coverslipper</td>
			<td>Sakuraus</td>
			<td>DRS-Prisma-P-JCS&#38;Film-JC2</td>
			<td></td>
		</tr>
		<tr>
			<td>CD163</td>
			<td>GrowGn Biotechnology</td>
			<td>NCL-L-CD163</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1a</td>
			<td>Gene Tech</td>
			<td>GM357129</td>
			<td></td>
		</tr>
		<tr>
			<td>CD56</td>
			<td>Gene Tech</td>
			<td>GT200529</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8</td>
			<td>Novocastra</td>
			<td>NCL-L-CD8-4B11</td>
			<td></td>
		</tr>
		<tr>
			<td>Dehydrator</td>
			<td>Thermo Fisher</td>
			<td>Excelsior ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital pathology and</td>
			<td>Indica labs</td>
			<td>HALO</td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3</td>
			<td>YILIFANG biological</td>
			<td>14-477-82</td>
			<td></td>
		</tr>
		<tr>
			<td>IHC stainer</td>
			<td>Leica</td>
			<td>BOND III</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis platform</td>
			<td>Indica labs</td>
			<td>HALO</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Olympus life science</td>
			<td>VS200</td>
			<td></td>
		</tr>
	</tbody>
</table>

platform for quantitative detection of endometrial immune cells based on immunohistochemistry and digital image analysis

为评估复发性流产（RM）患者的子宫内膜免疫微环境，开发并验证了数字免疫组化图像分析平台，用于定量分析黄体中期子宫内膜免疫细胞。所有子宫内膜样本均在月经周期的黄体中期采集。将石蜡包埋的子宫内膜组织切片成4 μm厚的载玻片，进行免疫组化（IHC）染色检测子宫内膜免疫细胞，包括CD56+ uNK细胞、Foxp3+ Tregs、CD163+ M2巨噬细胞、CD1a+ DCs和CD8+ T细胞。使用数字玻片扫描仪对全景玻片进行扫描，并使用商业图像分析系统进行定量分析。子宫内膜免疫细胞的百分比是通过除以总子宫内膜细胞中的免疫细胞数来计算的。使用商用图像分析系统，可以轻松准确地分析传统图像分析难以或不可能分析的子宫内膜免疫细胞的定量评估。该方法可用于定量表征子宫内膜微环境，包括免疫细胞之间的相互作用，以及不同生殖失败患者的异质性。子宫内膜免疫细胞定量评估平台对RM患者的诊断和治疗具有重要的临床意义。

为了定量评估子宫内膜免疫细胞，减少人为操作失误带来的不稳定性，利用免疫组化自动检测和数字定量评价系统，建立了子宫内膜免疫细胞数字化定量分析平台。建立免疫组化图像分析平台，定量分析复发性流产（RM）患者植入窗口期的子宫内膜免疫细胞。所有子宫内膜组织均在月经周期的黄体中期收集。将石蜡包埋的子宫内膜组织切片成4 μm厚的载玻片，进行IHC染色检测子宫内膜免疫细胞，包括CD56+uNK细胞、Foxp3+Tregs、CD163+M2巨噬细胞、CD1a+DCs、CD8+T细胞。使用数字玻片扫描仪对全景玻片进行扫描，并使用数字图像分析系统进行定量分析。为了计算子宫内膜免疫细胞的百分比，将免疫细胞的数量除以子宫内膜细胞的总数。 （图 1 和图 2）。RM女性中不同子宫内膜免疫细胞的比例（N = 30）如表2所示。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65643/65643fig01.jpg"> 图1：黄体中期子宫内膜免疫细胞分析的工作流程示意图。在月经周期的黄体中期，即黄体生成素 （LH） 激增后 7-9 天进行样本采集。在室温下在10%中性缓冲福尔马林中固定4-6小时，然后包埋在石蜡中。黄体中期子宫内膜免疫细胞检测，包括CD56+uNK细胞、Foxp3+Tregs、CD68+巨噬细胞、CD163+M2巨噬细胞、CD1a+DCs和CD8+T细胞25,26。使用商用扫描仪扫描组织，并使用免疫组化图像分析系统进行定量分析。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65643/65643fig01large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65643/65643fig02.jpg"> 图 2：子宫内膜细胞的免疫染色，用于鉴定阳性免疫细胞。RM患者子宫内膜CD56+uNK细胞、Foxp3+Tregs、CD68+巨噬细胞、CD163+M2巨噬细胞、CD1a+DCs和CD8+T细胞的免疫染色。棕色代表阳性免疫细胞，蓝色代表细胞核。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65643/65643fig02large.jpg" target="_blank">请点击这里查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>一抗</td>
			<td>克隆</td>
			<td>稀释</td>
		</tr><tr><td>CD56型</td>
			<td>123C3型</td>
			<td>1/800</td>
		</tr><tr><td>狐狸3</td>
			<td>型号：236A/E7</td>
			<td>1/100</td>
		</tr><tr><td>CD163型</td>
			<td>10天6</td>
			<td>1/1200</td>
		</tr><tr><td>CD1a型</td>
			<td>10</td>
			<td>1/200</td>
		</tr><tr><td>CD8型</td>
			<td>4B11型</td>
			<td>1/300</td>
		</tr></tbody></table>表1：免疫组化染色过程中使用的一抗。 下表显示了一抗的克隆和稀释度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>免疫标志物</td>
			<td>中位数 （%）</td>
			<td>最小值 （%）</td>
			<td>最大值 （%）</td>
			<td>平均值 （%）</td>
			<td>第 5 个百分位 （%）</td>
			<td>第 95 个百分位 （%）</td>
		</tr><tr><td>CD56型</td>
			<td>4.83</td>
			<td>1.8</td>
			<td>16.76</td>
			<td>6.03</td>
			<td>2.04</td>
			<td>13.63</td>
		</tr><tr><td>狐狸3</td>
			<td>0.05</td>
			<td>0.02</td>
			<td>0.12</td>
			<td>0.06</td>
			<td>0.02</td>
			<td>0.11</td>
		</tr><tr><td>CD68型</td>
			<td>0.78</td>
			<td>0.37</td>
			<td>3.62</td>
			<td>0.99</td>
			<td>0.44</td>
			<td>2.67</td>
		</tr><tr><td>CD163型</td>
			<td>0.84</td>
			<td>0.35</td>
			<td>2.38</td>
			<td>0.93</td>
			<td>0.38</td>
			<td>2.13</td>
		</tr><tr><td>CD1a型</td>
			<td>0.04</td>
			<td>0.01</td>
			<td>0.11</td>
			<td>0.05</td>
			<td>0.01</td>
			<td>0.1</td>
		</tr><tr><td>CD8型</td>
			<td>1.69</td>
			<td>0.76</td>
			<td>4.1</td>
			<td>1.85</td>
			<td>0.81</td>
			<td>3.72</td>
		</tr></tbody></table>表 2：RM 女性中各种子宫内膜免疫细胞的百分比。 采用免疫组化图像分析平台计算RM患者CD56+uNK细胞、Foxp3+Tregs、CD68+巨噬细胞、CD163+M2巨噬细胞、CD1a+DCs和CD8+T细胞的百分比。

Watch this Scientific Journal Video about Advancing Reproductive Immunology with a Protocol for the Quantitative Evaluation of Endometrial Immune Cells at JoVE.com

作者聚焦：通过子宫内膜免疫细胞定量评估方案推进生殖免疫学 

本文开发并验证了数字免疫组化图像分析平台，用于定量分析复发性流产患者在植入窗口期的子宫内膜免疫细胞。

基于免疫组化和数字图像分析的子宫内膜免疫细胞定量检测平台

To evaluate the endometrial immune microenvironment of patients with recurrent miscarriage (RM), a digital immunohistochemistry image analysis platform ...

目前，将分析稳定在一定范围内具有挑战性。保持实验的质量控制也很困难。我们的研究可以帮助免疫内膜敏感性异常的患者找到生殖衰竭的原因。子宫内膜内免疫细胞的局部比例可以稳定、反复地检测。本研究结果可促进反复着床失败患者子宫内膜敏感性评估的研究进展。

platform-for-quantitative-detection-endometrial-immune-cells-based-on

Nanyang Technological University

Research

JoVE Journal

Biology

Platform for Quantitative Detection of Endometrial Immune Cells Based on Immunohistochemistry and Digital Image Analysis

1.1K 次观看.  Shenzhen Zhongshan Urology Hospital. 目前，将分析稳定在一定范围内具有挑战性。保持实验的质量控制也很困难。我们的研究可以帮助免疫内膜敏感性异常的患者找到生殖衰竭的原因。子宫内膜内免疫细胞的局部比例可以稳定、反复地检测。本研究结果可促进反复着床失败患者子宫内膜敏感性评估的研究进展。

视频: 作者聚焦：通过子宫内膜免疫细胞定量评估方案推进生殖免疫学 

A Quantitative Fitness Analysis Workflow

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. QFA can be applied to focused observations of single cultures but is most useful for genome-wide genetic interaction or drug screens investigating up to thousands of independent cultures. The central experimental method is the inoculation of independent, dilute liquid microbial cultures onto solid agar plates which are incubated and regularly photographed. Photographs from each time-point are analyzed, producing quantitative cell density estimates, which are used to construct growth curves, allowing quantitative fitness measures to be derived. Culture fitnesses can be compared to quantify and rank genetic interaction strengths or drug sensitivities. The effect on culture fitness of any treatments added into substrate agar (e.g. small molecules, antibiotics or nutrients) or applied to plates externally (e.g. UV irradiation, temperature) can be quantified by QFA.
The QFA workflow produces growth rate estimates analogous to those obtained by spectrophotometric measurement of parallel liquid cultures in 96-well or 200-well plate readers. Importantly, QFA has significantly higher throughput compared with such methods. QFA cultures grow on a solid agar surface and are therefore well aerated during growth without the need for stirring or shaking.
QFA throughput is not as high as that of some Synthetic Genetic Array (SGA) screening methods5,6. However, since QFA cultures are heavily diluted before being inoculated onto agar, QFA can capture more complete growth curves, including exponential and saturation phases3. For example, growth curve observations allow culture doubling times to be estimated directly with high precision, as discussed previously1.
Here we present a specific QFA protocol applied to thousands of S. cerevisiae cultures which are automatically handled by robots during inoculation, incubation and imaging. Any of these automated steps can be replaced by an equivalent, manual procedure, with an associated reduction in throughput, and we also present a lower throughput manual protocol. The same QFA software tools can be applied to images captured in either workflow.
We have extensive experience applying QFA to cultures of the budding yeast S. cerevisiae but we expect that QFA will prove equally useful for examining cultures of the fission yeast S. pombe and bacterial cultures.

Quantitative Fitness Analysis (QFA) is a complementary series of experimental and computational methods for estimating microbial culture fitnesses. QFA estimates the effect of genetic mutations, drugs or other applied treatments on microbe growth. Experiments scaling from focussed analysis of single cultures to thousands of parallel cultures can be designed.

定量健身分析工作流程

Quantitative Fitness Analysis (QFA) is an experimental and computational workflow for comparing fitnesses of microbial cultures grown in parallel1,2,3,4. ...

科研

生物学

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

多蛋白复合物的比较分析新方法基于<sup&gt; 15</sup&gt;Ñ代谢标记和定量质谱

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

利用荧光标记的纳米粒子检测的小鼠，人与猪源活细胞内的基因表达

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Using Digital Image Correlation to Characterize Local Strains on Vascular Tissue Specimens

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and product development. Stress-strain relationships are typically obtained from mechanical testing data to enable comparative assessment among samples and in some cases identification of constitutive mechanical properties. However, errors may be introduced through the use of average strain measures, as significant heterogeneity in the strain field may result from geometrical non-uniformity of the sample and stress concentrations induced by mounting/gripping of soft tissues within the test system. When strain field heterogeneity is significant, accurate assessment of the sample mechanical response requires measurement of local strains. This study demonstrates a novel biomechanical testing protocol for calculating local surface strains using a mechanical testing device coupled with a high resolution camera and a digital image correlation technique. A series of sample surface images are acquired and then analyzed to quantify the local surface strain of a vascular tissue specimen subjected to ramped uniaxial loading. This approach can improve accuracy in experimental vascular biomechanics and has potential for broader use among other native soft tissues, engineered soft tissues, and soft hydrogel/polymeric materials. In the video, we demonstrate how to set up the system components and perform a complete experiment on native vascular tissue.

We describe the use of digital image correlation to characterize the local surface strain field on vascular tissue samples subjected to uniaxial tensile testing. These measurements facilitate precise quantification of the sample mechanical response and the generation of constitutive stress-strain relations.

利用数字图像相关表征的血管组织标本本地菌株

Characterization of the mechanical behavior of biological and engineered soft tissues is a central component of fundamental biomedical research and ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

胞嘧啶修饰形式使用敏感免疫组化检测

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

利用两色单分子荧光在原位杂交中Escherichia coli探索大肠杆菌中空间和时间中的 mRNA 动力学

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

基于免疫组化和数字图像分析的子宫内膜免疫细胞定量检测平台

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