这项工作得到了国家自然科学基金（82060341,81560304）和海南省院士创新平台科研项目（YSPTZX202134）的支持。

该研究已获得海南医科大学伦理委员会的批准。本研究中使用的组织样本不被视为涉及人类受试者的研究。这些标本是出于法医病理诊断的目的而获得的，其余的用于本实验中硅藻DNA的提取。研究人员无法轻易识别个人以获得相关利益相关者的知情同意。
注：为保证本实验中报告的研究方法的普遍适用性，本实验基本遵循所用试剂盒的操作说明，仅修改了部分步骤。本实验中使用的水样是从实验室附近的池塘中随机采集的（补充图1A）。在该实验中，确认溺水尸体的肺组织为研究组织，以证明提取方案（补充图1B）。在法医实践中，有时还需要使用溺水尸体的其他器官和组织（如肝、脾、肾、骨髓等）来提取硅藻DNA，这需要对这种实验方法进行微小的相应改进，这将在实验的相应章节中解释。该实验中使用的肺组织样本来自法医案件中明显淹死的尸体。已经进行了形态学测试，以证明肺组织含有硅藻（补充图2）。
1.样品的预处理
<ol><li>水样的预处理<ol><li>将 10 mL 水样放入离心管中，并以 13,400 x g 离心 5 分钟。用移液管小心弃去 9.8 mL 上清液，并将剩余在底部的约 200 μL 富集硅藻水样品转移到 2 mL 离心管中。 注意：可以先进行预实验，如果 10 mL 水样不足以富集，可以增加初始量。</li>
	</ol></li>
	<li>组织样本的预处理<ol><li>从溺水的尸体中取出0.5克肺边缘组织，将肺组织完全切碎或研磨，直到它变得浑浊。防止外源硅藻污染是这一步的核心。用剪刀反复将组织切成小块。</li>
	</ol></li>
</ol>2. DNA提取
注意：所有离心步骤均在室温下完成。使用离心力为14,500 x g的台式离心机;在实验开始之前，需要准备预热到70°C的水浴（或金属浴）。所有步骤必须严格遵循无菌操作原则。
<ol><li>水样和组织的组装 注意：水样和组织硅藻DNA提取方法相同。<ol><li>将玻璃珠加入含有预处理水样的 2 mL 离心管中。倒置 10 倍至 15 倍以充分混合。添加的玻璃珠由大小玻璃珠以1：1的质量比混合而成。大玻璃珠的直径为 1.5-2.0 毫米，小玻璃珠的直径为 0.4-0.6 毫米。</li>
		<li>取 0.5 g 切碎的组织，如上所述将玻璃珠加入含有组织的 2 mL 离心管中。倒置 10 倍至 15 倍混合。</li>
	</ol></li>
	<li>向试管中加入 40 μL 蛋白酶 K （20 mg/mL）。在室温下放置 15 分钟，在此期间，倒置，每 3 分钟混合 10 次。 注意：如果组织没有完全消化，可以适当增加蛋白酶K的量，直到溶液变得透明。</li>
	<li>向离心管中加入200μL结合缓冲液，立即涡旋，混合4分钟（振荡混合频率：3000rpm）。 注意：在此步骤中，应严格控制振荡强度和时间，以确保足够的混合强度和时间。</li>
	<li>将离心管置于70°C水浴中10分钟，溶液变得澄清。</li>
	<li>向离心管中加入100μL异丙醇，涡旋并混合15秒，此时可能出现絮状沉淀。 注意：在上述操作步骤中，以适当的强度充分混合非常重要，但应避免剧烈摇晃，以防止DNA剪切。</li>
	<li>将上一步得到的溶液与絮凝剂沉淀物一起放入吸附柱中（吸附柱置于收集管中）。吸附柱长度为3.0厘米，直径为1.0厘米，吸附基体为硅基膜。</li>
	<li>以8,000× g 离心30秒，弃去收集管中的废液，并将吸附柱放回收集管中。</li>
	<li>向吸附柱中加入 500 μL 抑制剂去除缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。</li>
	<li>向吸附柱中加入700μL洗涤缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。 注意： 首次使用前，将指定量的无水乙醇加入洗涤缓冲液瓶中。</li>
	<li>向吸附柱中加入 500 μL 洗涤缓冲液。以 13,400 x g 离心 30 秒，然后丢弃收集管中的废液。</li>
	<li>将吸附柱放回空的收集管中。以14,500× g 离心2分钟，并尽可能除去洗涤缓冲液，以避免洗涤缓冲液中残留的乙醇抑制下游反应。</li>
	<li>取出吸附柱，放入干净的离心管中。向吸附膜的中间部分加入 100 μL 洗脱缓冲液。</li>
	<li>将吸附柱置于室温下3-5分钟，并以13,400× g 离心1分钟。</li>
	<li>将上一步得到的溶液再次加入离心吸附柱中。将离心吸附柱置于室温下2分钟，并以13,400× g 离心1分钟。 注：洗脱缓冲液应在70°C水浴中预热约10分钟。洗脱体积应不小于50μL;否则，DNA产量将降低。</li>
	<li>将提取的硅藻DNA储存在2-8°C以备将来使用。如果要长时间储存DNA溶液，请将其储存在-20°C。</li>
</ol>3. PCR检测
注意：由于法医样本中硅藻的含量通常很低，溺水尸体的组织样本提取物也可能含有不同程度的组织和器官（例如本实验中的肺）及其自身的DNA。因此，直接检测DNA提取物中的总DNA并不能反映硅藻DNA提取的情况。本实验选择硅藻特异性引物，采用PCR产物对提取物中的硅藻DNA提取情况进行评价。产物可采用琼脂糖凝胶电泳进行观察分析，也可采用实时荧光定量PCR熔解曲线进行分析，灵敏度更高。
<ol><li>加入 2 μL 从水样和组织中提取的 DNA 作为模板。选择以下两种方法之一进行检查。</li>
	<li>常规PCR检测<ol><li>使用可特异性扩增硅藻 18S rDNA 片段的引物22 。具体请参见 表1。</li>
		<li>根据引物的特性建立PCR反应体系和扩增条件。具体请参见 表2。</li>
		<li>在2%琼脂糖凝胶上运行PCR扩增产物。使用凝胶成像仪观察和分析成像。</li>
	</ol></li>
	<li>荧光定量PCR检测<ol><li>使用上述可特异性扩增硅藻18S rDNA片段的引物，制备实时荧光定量PCR反应体系。具体请参见 表3。</li>
		<li>进行PCR扩增并分析获得的扩增曲线和Ct值。同时，设置程序，通过荧光定量PCR熔融曲线技术，将扩增产物的双链逐渐熔融成单链，然后对得到的熔解曲线进行分析。</li>
	</ol></li>
</ol>

使用改良试剂盒进行经济的硅藻 DNA 提取

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硅藻细胞受到坚硬的硅质细胞壁17的保护，必须破坏这种结构才能提取硅藻DNA。普通试剂盒不易破坏硅藻的硅质外壳;因此很难成功提取硅藻DNA21。我们的实验室改进了最常用的血液DNA提取试剂盒，在硅藻提取过程中添加了不同直径和不同质量比的玻璃珠。同时进行涡旋振荡，可充分打破硅藻壳，提高硅藻DNA提取效果。如上一份报告21所示，玻璃...

在法医实践中，确定尸体是在水中溺水还是死后被扔进水里，对于案件的妥善解决至关重要1.这也是法医学实践中亟待解决的难题之一2.硅藻在自然环境中（尤其是在水中）含量丰富3,4。在溺水过程中，由于缺氧和应激反应，患者会出现剧烈的呼吸运动，吸入大量的溺水液。因此，水中的硅藻随溺水液进入肺部，一些硅藻可以通过肺泡-毛细血管屏障进入血液循环，并随着血流扩散到内脏器官5,6。在肺、肝和骨髓等内部组织和器官中检测到硅藻是死前溺水的有力证据 7,8。目前，法医硅藻测试主要基于形态学测试方法。在对组织进行一系列预消化后，在显微镜下对未消化的硅藻进行形态定性和定量估计。在此期间，需要使用危险且对环境不友好的试剂，例如硝酸。这个过程非常耗时，需要研究人员具有扎实的分类学专业知识和丰富的经验。这些都给法医人员带来了一定的挑战9.硅藻DNA检测技术是近年来发展起来的一种用于硅藻检测的新技术10,11,12。该技术通过分析硅藻的特定DNA序列组成来实现硅藻的物种鉴定13,14。PCR技术和测序技术是常用的技术方法，但它们的基础是从硅藻中成功提取DNA。然而，硅藻具有不同于其他生物的特殊结构，使其DNA提取技术也不同。
硅藻的细胞壁具有高度的硅化程度，其主要成分是二氧化硅15,16,17。硅质细胞壁非常坚硬，在提取DNA之前必须将其破坏。普通的DNA提取试剂盒通常难以直接用于硅藻DNA的提取，因为它们不能破坏硅藻的硅质外壳18。因此，破坏硅藻的硅质外壳是提取硅藻DNA需要解决的关键技术问题之一。
同时，由于法医研究样本中所含的硅藻数量，无论是水样还是溺水尸体的器官和组织，往往都是有限的，因此有必要富集硅藻。富集的本质是物质的分离。在尝试将硅藻聚集在一起时，尽量减少其他材料成分（干扰成分）的含量。在法医工作中，实验室经常使用离心或膜过滤富集方法来分离硅藻细胞19。然而，由于真空泵设备应用不广，膜富集法在普通的初级法医实验室中并不常用。因此，离心法仍然是法医实验室20中常用的硅藻富集法。
从硅藻中提取DNA目前主要用于法医实践，其应用存在很大的局限性。目前，市场上用于法医学的硅藻DNA提取试剂盒很少，而且价格普遍昂贵21。本文提供了一种改进的硅藻DNA提取方法，使硅藻DNA提取变得简单、方便且具有成本效益。这增加了后续硅藻分子生物学检测的应用，可以通过硅藻检测更好地解决法医溺水相关问题。该方法通过添加玻璃珠并设置适当的涡旋时间来破坏硅藻的硅质细胞壁。通过这种方式，蛋白酶 K 和结合溶液迅速裂解细胞并使细胞中的各种酶失活。基因组DNA在吸附柱的基质膜中被吸收，最后被洗脱缓冲液洗脱。这种改进的全血基因提取试剂盒提高了血液试剂盒在法医检验材料中的硅藻DNA提取效果，降低了法医实践中硅藻DNA提取的成本，可以更好地应用于基层法医研究。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Binding Buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>rapidly lysing cells</td>
		</tr>
		<tr>
			<td>ChemoHS qPCR Mix</td>
			<td>Monad</td>
			<td>00007547-120506</td>
			<td>qPCR Mix</td>
		</tr>
		<tr>
			<td>D2000 DNA ladder</td>
			<td>Real-Times(Beijing) Biotechnology</td>
			<td>RTM415</td>
			<td>Measure the position of electrophoretic bands</td>
		</tr>
		<tr>
			<td>D512</td>
			<td>Taihe Biotechnology</td>
			<td>TW21109196</td>
			<td>forword primer</td>
		</tr>
		<tr>
			<td>D978</td>
			<td>Taihe Biotechnology</td>
			<td>TW21109197</td>
			<td>reverse primer</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>A low-salt elution buffer washes off the DNA</td>
		</tr>
		<tr>
			<td>Glass bead</td>
			<td>Yingxu Chemical Machinery(Shanghai)&#160;</td>
			<td>70181000</td>
			<td>Special glass beads for dispersing and grinding</td>
		</tr>
		<tr>
			<td>Import adsorption column</td>
			<td>BioTeke</td>
			<td>B2008006022</td>
			<td>Adsorption column with silica matrix membrane</td>
		</tr>
		<tr>
			<td>Inhibitor Removal Buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Removal of Inhibitors in DNA Extraction</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Precipitate or isolate DNA</td>
		</tr>
		<tr>
			<td>MIX-30S Mini Mixer</td>
			<td>Miulab</td>
			<td>MUC881206</td>
			<td>oscillatory action</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Inactivation of intracellular nucleases and other proteins</td>
		</tr>
		<tr>
			<td>Rotor-Gene Q 5plex HRM</td>
			<td>Qiagen</td>
			<td>R1116175</td>
			<td>real-time fluorescence quantification PCR</td>
		</tr>
		<tr>
			<td>Speed Micro-Centrifuge</td>
			<td>Scilogex</td>
			<td>9013001121</td>
			<td>centrifuge</td>
		</tr>
		<tr>
			<td>Tanon 3500R Gel Imager</td>
			<td>Tanon</td>
			<td>16T5553R-455</td>
			<td>gel imaging</td>
		</tr>
		<tr>
			<td>Taq Mix Pro</td>
			<td>Monad</td>
			<td>00007808-140534</td>
			<td>PCR Mix</td>
		</tr>
		<tr>
			<td>Thermo Cycler</td>
			<td>Zhuhai Hema</td>
			<td>VRB020A</td>
			<td>ordinary PCR</td>
		</tr>
		<tr>
			<td>Wash Buffer</td>
			<td>BioTeke</td>
			<td>B010006022</td>
			<td>Remove impurities such as cell metabolites</td>
		</tr>
	</tbody>
</table>

extraction of diatom dna from water samples and tissues

硅藻测试是法医实践中确定尸体是否溺水、推断溺水地点的重要辅助手段。硅藻试验也是环境和浮游生物领域的重要研究内容。以硅藻DNA为主要研究对象的硅藻分子生物学检测技术是一种新的硅藻检测方法。硅藻DNA提取是硅藻分子检测的基础。目前，常用用于硅藻DNA提取的试剂盒价格昂贵，增加了开展相关研究的成本。本实验室对通用全血基因组DNA快速提取试剂盒进行了改进，获得了满意的硅藻DNA提取效果，从而为相关研究提供了一种经济实惠的基于玻璃珠的DNA提取解决方案。使用该方案提取的硅藻DNA可以满足许多下游应用，例如PCR和测序。

由于目前使用的DNA提取方法提取的DNA溶液中含有来自样品中不同来源的所有DNA成分，因此通过该协议获得的DNA也不例外。因此，DNA溶液不仅仅是硅藻基因组DNA的溶液。通过查阅文献22,23,24选择可特异性扩增硅藻18S rDNA片段的引物。采用NCBI-Blast Primer对引物进行验证，结果表明，18S rDNA的正向引物D512和反向引物D978是藻类特异性引?...

Watch this Scientific Journal Video about Diatom Testing for Forensic Drowning Examination at JoVE.com

作者聚焦： 用于法医溺水检查的硅藻检测 

本文介绍了使用改良的通用 DNA 提取试剂盒提取硅藻 DNA 的方案。

从水样和组织中提取硅藻DNA

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

我们的研究主要集中在溺水的话题上。我们想回答在水中发现的尸体是否死于溺水的问题，以及回答另一个人落入水中的另一个问题。这意味着要知道溺水的位置。与其他技术相比，我们提供了一种简单、方便、经济的硅藻DNA提取方案，我们可以更好地用于法医研究，特别是在基本的法医实践中。我们的实验室将根据水中的模式，如死亡原因、死后间隔等，进一步关注溺水相关问题。此外，我们还通过法医昆虫学和热带法医中的一些特殊现象进行研究。

extraction-of-diatom-dna-from-water-samples-and-tissues

Research

JoVE Journal

Medicine

Extraction of Diatom DNA from Water Samples and Tissues

1.7K 次观看.  Hainan Medical University, Hainan Provincial Academician Workstation (tropical forensic medicine). 我们的研究主要集中在溺水的话题上。我们想回答在水中发现的尸体是否死于溺水的问题，以及回答另一个人落入水中的另一个问题。这意味着要知道溺水的位置。与其他技术相比，我们提供了一种简单、方便、经济的硅藻DNA提取方案，我们可以更好地用于法医研究，特别是在基本的法医实践中。我们的实验室将根据水中的模式，如死亡原因、死后间隔等，进一步关注?...

视频: 作者聚焦： 用于法医溺水检查的硅藻检测 

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

科研

医学

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

快速分馏和全血的成分的分离样品中从社区为基础的设置获得

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

临床样品RNA病毒的无偏深度测序

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

猪生物医学模型生物库组织样品的取样策略与处理

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial infarction, arrhythmia, atherosclerosis, cardiogenic pulmonary edema, pulmonary embolism, and cerebral embolism. At the same time, it is urgent to break through the continuous heart blood sampling technique in live rats to evaluate the effectiveness of distinctive ethnic medicine therapy. In this study, a blood microdialysis probe was implanted in the right jugular vein of rats in a precise and noninvasive surgical procedure. Cardiac blood samples were then collected at a rate of 2.87 nL/min to 2.98 mL/min by connecting to an online microdialysis sample collection system. Even more momentously, the acquired blood samples can temporarily be stored in microdialysis containers at 4 &#176;C. The microdialysis-based online continuous blood collection program from rat heart has greatly guaranteed the quality of blood samples, advancing and invigorating the scientific rationality of the research on systemic cardiovascular diseases and evaluating ethnomedicine therapy from the perspective of hematology.

Dynamic Continuous Blood Extraction from Rat Heart via Noninvasive Microdialysis Technique

The present protocol describes a simple and efficient method for the real-time and dynamic collection of rat heart blood using the microdialysis technique.

无创微透析 技术从 大鼠心脏动态连续提取血液

Dynamic analysis of blood components is of great importance in understanding cardiovascular diseases and their related diseases, such as myocardial ...

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 &#176;C and 4 &#176;C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.

The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.

从羊膜中提取Lumican及其储存温度的测定

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen ...

过敏性鼻炎大鼠模型中鼻组织的多色免疫荧光

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

作者聚焦:利用多色免疫荧光推进过敏性鼻炎研究

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

通过多色免疫测定法对过敏性鼻炎大鼠鼻粘膜组织切片进行免疫荧光标记

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...