We would like to acknowledge our colleague Dr. Jalaiah Varikooty who provided insight and expertise on the topic of the lid wiper, that greatly assisted this research.

伦理学声明:在此之前，从人类受试者采集细胞，知情同意，并且必须获得伦理委员会批准。 1.准备染色注意:在实验当天准备污点。 <ol><li>在15ml离心管5微升乙锭（或4μM）和5μl钙黄绿素AM（或4​​μM）添加到2.5毫升磷酸盐缓冲盐水（PBS）;混合。包装在铝箔在室温避光和存储屏蔽直到使用。 </li></ol> 2.收集细胞<ol><li>从包装中取出细胞培养插入和标签为每个眼睑进行采样。细胞收集的关于使用永久标记在膜的塑料保持器的侧标记定向。行为裂隙灯检查在中等倍率（约20倍），以确认该区域的适当的健康接受的IC。 </li><li>在EA的低结膜囊分配局部麻醉剂（0.5％丙美卡因盐酸盐）一滴CH眼睛和指示参与者保持闭眼一分钟。 </li><li>埃弗特上眼睑和由睫毛的地方举行;避免与盖刮水器区域中的任何接触。 可选的:一个次级研究者的帮助可能需要执行此步骤。 </li><li>垂直应用膜到盖刮水器区域的最小压力的中心部分。 </li><li> 5秒 - 到位3握住膜。观察该膜中的接触面积变得半透明。在这一点上，轻轻地除去膜并允许参与者的盖子为"翻转"回原位。 </li><li>及时的PBS一滴施加到样品，以防止其干燥。膜将变成半透明的，并且在任何时候不应该把不透明的，因为这表明干燥。有研究者二次用样品的处理（第3节）立即开始。 </li><li>最后进行眼部检查，以确保结膜的完整性。免除眼部润滑剂和指导参与者避免揉眼睛直到麻醉效果消退（约15分钟）。 </li></ol> 3.样品处理<ol><li>用微型剪刀，切割膜中一半（一半用于每个下游分析的），垂直通过细胞收集的中间。沿着一个膜一半将它从塑料保持器分离的外缘切割;避免与在膜上收集的细胞，包括确保所述膜不折叠到自身相互作用。 </li><li>从持有者完全分离前安全用镊子膜。地方膜到含有500μl的95％（V / V）乙醇和离开处理（第4.2节）之前定为至少20分钟至最多2小时的标记为2毫升离心管中。 </li><li>膜分离立即下半年（如3.2节所述）和第4.1及时进行。 </li></ol> 4.样品染色<ol><li> ImmunocytochemiCAL染色<ol><li>小心将膜与收集面朝下跌35毫米的玻璃底培养皿;确保膜是平的。 </li><li>吸取20微升第1条组装到膜免疫细胞化学染色组成。如果样品缭绕，使用一次性吸头推其边缘下来仔细扁平化膜。避免细胞收集区接触。 </li><li>轻轻盖上盖玻片膜。避免样品的不必要的移动。盖上盖子的菜，并与周围的边缘实验室薄膜密封。不会阻碍培养皿（顶部和底部）和样品的可视性的透明性。与实验室标志的标签。 </li><li>与成像（5.1节）立即着手，然后返回到第4.2节继续与膜的另一半的细胞学染色。 </li></ol></li><li> 细胞学染色 <ol><li>通过缓慢加入500微升的蒸馏水到管ü逐渐水合膜3.4节的sed进行固定。内容抽吸数次进入枪头混合，然后删除内容。 </li><li>加入500微升的蒸馏水。让我们站在了几秒钟，然后取出。加入500μl阿尔新蓝染色，离开3分钟。除去污点并进行连续3 500微升蒸馏水水冲洗或直到液体冲洗清晰。 </li><li>加入500μl苏木＃1色斑，离开3分钟。除去污点并进行连续三个500微升蒸馏水水冲洗或直到液体冲洗清晰。通过吸取透明液体脱水（4.2.1节反向）。 </li><li>加入500μl巴氏OG-6染色的，离开3分钟。去除污点和执行一个500μl的95％（体积/体积）乙醇漂洗。 </li><li>加入500μl巴氏EA-65染色，并留下3分钟。除去污渍，并执行连续3次500μl的95％（体积/体积）乙醇漂洗。 </li><li>充分利用脱水连续三个500微升100％乙醇漂洗。使用TWEezers，去除溶液样品;避免接触细胞采集领域。 </li><li>放在载玻片膜;确保细胞收集行是平行或垂直于成像期间更容易对准滑动余量。 </li><li>应用100％的乙醇的下降，并与玻璃滑移盖。立即着手与成像（第5.2节）。 </li></ol></li></ol> 5.样品成像<ol><li> 成像荧光免疫细胞化学染色剂 <ol><li>使用共聚焦激光扫描显微镜（CLSM）11或荧光显微镜12中 ，配备有数码彩色摄像机，连接到运行的图像获取软件的计算机。由于样品被包含培养皿内，在倒置显微镜可能是必要的。 </li><li>设置显微镜11,12根据乙锭同型二聚体-1（六百十七分之五百二十八毫微米实施例/ EM最大值在DNA的存在下）和钙黄绿素AM的吸收和发射光谱（五百十七分之四百九十四纳米ËX / EM最大值）。 </li><li>选择显微镜的放大倍率最低（ 例如，2.5X目标），并激活数字成像系统;观察电脑显示器上显示的样本。 </li><li>检查感兴趣细胞特征的样本。 </li><li>选择需要的显微镜的放大倍率，并使用图像处理软件采集图像。 </li><li>无论是在本地显微镜文件格式或未压缩的文件格式（ 例如 ，* .RAW，* .TIFF）保存文件。 </li></ol></li><li> 成像细胞学渍 <ol><li>使用不带任何过滤器标准实验室亮场显微镜，配备数字彩色摄像头，连接到运行图像采集软件的计算机。 </li><li>地方和锁定载玻片（从步骤4.2.8）在显微镜阶段。根据需要再水合用100％乙醇样品，在整个成像过程。 </li><li>选择显微镜的放大倍率最低（ 例如 ，2.5X目标），并激活次Ë数字成像系统;观察电脑显示器上显示的样本。 </li><li>确保细胞收集与显微镜载物台控制的X或Y轴对齐。如果需要的话，通过除去盖玻片并用移液管尖端或镊子转动样品调整。 </li><li>使用成像软件，用最低的显微镜放大倍数整个样本的捕获图像（多个）。 </li><li>切换到中等显微镜放大倍率（ 例如，10 - 20X的目标），并调整显微镜光源和软件成像参数（曝光，对比度，白平衡等 ），并禁止他们的自动化软件测光。保存以备将来使用这些设置。 </li><li>确定扫描（ 例如 ，左上角和细胞收集的右下角）的开始和结束点。 </li><li>使用成像软件中，起始和结束点的范围内的全部细胞收集区的捕获图像（多个）。确保20％的侧到侧和顶部至底部重叠betwEEN所有相邻图像。 </li><li>使用本机显微镜文件格式或未压缩的文件格式（ 例如 ，* .RAW，* .TIFF）保存文件。 </li><li>使用所选的自动拼接软件（ 例如，Adobe公司的Photoshop Elements）生成最终的全景图像。使用），在步骤5.2.5拍摄参考图像，以确认自动缝合的准确性最终影像中。 </li></ol></li></ol>

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The authors have no relevant funding or conflicts to disclose.

印象细胞学检查是在球结膜通常进行的，使用混合纤维素酯膜。样品切成尺寸从散装材料片，消毒，使用镊子结膜表面施加和除去。使用相同的膜材料，活塞控制的IC器件最近已设计，以配合球结膜的表面几何形状，和维护应用程序13之间是一致的压力。这些方法是低效的窄，突出地弯曲的盖刮水器区域（ 见图1）。此外，混合的纤维素酯膜不提供对设置必要的透明度，其中亮...

人的上眼睑周围执行每天1万闪烁，只用1 - 2 mm窄结膜区域反对眼球。由于眨眼在其擦拭动作，睑缘的这一部分被称之为"盖雨刮器"区域2。假定摩​​擦惯常闪烁期间在该区域增加，由于该盖抹遍布全球。这可能在眼睛舒适一个显著的作用。隐形眼镜配戴者特别是经验眼部不适，这是主要的原因停止配戴镜片3之一。当接触透镜被放置在眼睛上，透镜材料和眼表面之间的摩擦系数已经显示出改变4。有证据表明，干症状可能与此改变的摩擦2,5,6。 此关联，也可以在临床上观察到的现象的反射，尤其是盖的Wi每上皮病变（LWE），也称为上盖余量染色（ULMS）6。 LWE是对眼有刺激性的早期征兆和干眼病的潜在指标。据观察，并通过上，下眼睑边缘区域的活体染色测定。虽然这个分级系统2及其临床有效性（即与主观症状相关性）仍在争论，眼部不适仍然为临床医师和科学家都一个难题，以及最重要的是，对于患者的不便。 最新的，鲜为人知的是，临床相关的变化盖子雨刮区7的（子）细胞的解剖和生理，只有一个报告中运用的方法细胞学8。印象细胞学检查（IC）已雇用了超过40年由膜9,10的应用程序来收集细胞延髓或睑结膜上皮表面。取出后，贴壁细胞经历Çytological染色和显微成像。由于在盖刮水器区域的解剖和细胞的差异，这种技术需要优化。

<table border="0" cellpadding="0" cellspacing="0" width="753">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:349px;">Alcian Blue, 1% in 3% Acetic Acid</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">B8438-250ML</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hematoxylin, Gill 1</td>
			<td style="width:119px;">Sigma</td>
			<td>GHS116-500ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, OG-6</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40116-500 ml</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Papanicolaou stain, Modified EA</td>
			<td style="width:119px;">Sigma</td>
			<td>HT40232-1L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Live/Dead Viability/Cytotoxicity Kit</td>
			<td>Life Technologies</td>
			<td>L-3224</td>
			<td>contains ethidium homodimer-1 and Calcein AM dyes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Alcaine (0.5% proparacaine hydrochloride)</td>
			<td>Alcon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Millicell Cell Culture Insert, 12&#160;mm, hydrophilic PTFE, 0.4&#160;&#181;m</td>
			<td>EMD Millipore</td>
			<td>PICM01250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">35 mm Glass Bottom Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
	</tbody>
</table>

impression cytology of the lid wiper area

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The optimization of a method of collecting, staining and imaging cells from the LW region using impression cytology (IC) is described in this study. Cells are collected from the inner surface of the upper eyelid of human subjects using hydrophilic polytetrafluoroethylene (PTFE) membranes, and stained with cytological dyes to reveal the presence of goblet cells, mucins, cell nuclei and various degrees of pre- and para-keratinization. Immunocytochemical dyes show cell esterase activity and compromised cell membranes by the use of a confocal scanning laser microscope. Up to 100 microscopic digital images are captured for each sample and stitched into a high-resolution, large scale image of the entire IC span. We demonstrate a higher sensitivity of IC than reported before, appropriate for identifying cellular morphologies and metabolic activity in the LW area. To our knowledge, this is the first time this selection of fluorescent dyes was used to image LW IC membranes. This protocol will be effective in future studies to reveal undocumented details of the LW area, such as assessing cellular particularities of contact lens wearers or patients with dry eye or lid wiper epitheliopathy.

跨越在塑料保持器细胞培养膜为从盖刮水器结膜上皮细胞的手持集合的方便工具。这种方法消除了通常用于制备和处理集成电路膜额外灭菌仪器的需要。 图1描绘了使用一个细胞培养插管在盖刮水器区的集成电路。我们优化的两种不同的染色方案互为补充，从盖雨刷区以新的方式表征上皮细胞。荧光免疫细胞化学染料揭示酯酶活性，细胞生存力的指标，和核酸...

Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

Impression Cytology of the Lid Wiper Area

We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort.

盖雨刮区的印象细胞学

Few reports on the cellular anatomy of the lid wiper (LW) area of the inner eyelid exist and only one report makes use of cytological methods. The ...

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10.3K 次观看.  University of Waterloo. We collected, stained and imaged cells from the conjunctiva of the inner eyelid margin of human subjects. By characterizing cell morphology and metabolic activity, this method may further our understanding of dry eye and the role that friction between the ocular surfaces may play in perceiving ocular discomfort. 

视频: Impression Cytology of the Lid Wiper Area

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

采用TaqMan从儿童凳诺如病毒的检测和Genogrouping一步法RT-PCR技术

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

科研

医学

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) 1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity3-8.
Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response11. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas12.
In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor's microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues4,8, but staining on paraffin-embedded slides had been a challenge until recently13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.

Immunohistochemical Staining of B7-H1 PD-L1 on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1 (PD-L1) and its binding to PD-1 provide a major tumor-induced immunosuppressive signal in the tumor&#x2019;s microenvironment. An immunohistochemical staining technique to characterize the expression and localization of B7-H1 in pancreatic adenocarcinoma is described here.

B7-H1（PD-L1）免疫组化染色石蜡的嵌入式幻灯片的胰腺癌组织

B7-H1/PD-L1, a member  of the B7 family of immune-regulatory cell-surface proteins, plays an important  role in the negative regulation of cell-mediated ...

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH的荧光成像隔离双心室工作离体兔心

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌:个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

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 ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

"以病人为像"肝癌原位同源小鼠模型的癌转移

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Induction of Ischemic Stroke and Ischemia-reperfusion in Mice Using the Middle Artery Occlusion Technique and Visualization of Infarct Area

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of stroke on tissue injury and to evaluate the effectiveness of therapeutic interventions, several experimental models in a variety of species were developed. They include complete global cerebral ischemia, incomplete global ischemia, focal cerebral ischemia, and multifocal cerebral ischemia. The model described in this protocol is based on the middle cerebral artery occlusion (MCAO) and is related to the focal ischemia category. This technique produces consistent focal ischemia in a strictly defined region of the hemisphere and is less invasive than other methods. The procedure described is performed on mice, given the availability of several genetic variants and the high number of tests standardized for mice to aid in the behavioral and neurodeficit evaluation.

We describe a mouse model of stroke induced by the occlusion of the middle cerebral artery using a silicone coated suture. The protocol can be applied to induce permanent occlusion or a temporary ischemia, followed by reperfusion.

缺血性中风和缺血再灌注小鼠的诱导使用梗塞区的中心动脉闭塞技术和可视化

Cerebrovascular disease is highly prevalent in the global population and encompasses several types of conditions, including stroke. To study the impact of ...

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This compound is currently under investigation as a PET tracer for demyelination. We recently described a novel chemical reaction to produce metafluorinated pyridines consisting of direct fluorination of a pyridine N-oxide and the utilization of this reaction for the radiochemical synthesis of [18F]3F4AP. In this article, we demonstrate how to produce this tracer using an automated synthesizer and an in-house made flow hydrogenation reactor. We also show the standard quality control procedures performed before releasing the radiotracer for preclinical animal imaging studies. This semi-automated procedure may serve as the basis for future production of [18F]3F4AP for clinical studies.

Automated Radiochemical Synthesis of [18F]3F4AP: A Novel PET Tracer for Imaging Demyelinating Diseases

We demonstrate the semi-automated radiochemical synthesis of [18F]3F4AP and quality control procedures.

自动放射化学合成[<sup&gt; 18</sup&gt; F] 3F4AP:一种用于成像脱髓鞘疾病的新型PET示踪剂

3-[18F]fluoro-4-aminopyridine, [18F]3F4AP, is a radiofluorinated analog of the FDA-approved drug for multiple sclerosis 4-aminopyridine (4AP). This ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

输卵管的管状细胞学作为卵巢癌早期检测的有前景的工具

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Accessing the Cytotoxicity and Cell Response to Biomaterials

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by several methods, but a high discrepancy exists between the approaches used, compromising the reproducibility and the comparison among the obtained results. In this paper, we propose a protocol to evaluate biomaterials cytotoxicity using soluble extracts, which we use for dental biomaterials. The extracts preparation is detailed, from pellets production to its extraction in a culture medium. The biomaterials cytotoxicity evaluation is based on metabolic activity using the MTT assay, cell viability using the Sulphorhodamine B (SBR) assay, cell death profile by flow cytometry, and cell morphology using May-Gr&#252;nwald Giemsa. Additional to cytotoxicity evaluation, a protocol to evaluate cell function is described based on the expression of specific markers assessed by immunocytochemistry and PCR. This protocol provides a comprehensive guide for biomaterials cytotoxicity and cellular effects evaluation, using the extracts methodology, in a reproducible and robust manner.

This methodology aims to evaluate biomaterial cytotoxicity through the preparation of soluble extracts, using viability assays and phenotypic analysis, including flow cytometry, RT-PCR, immunocytochemistry, and other cellular and molecular biology techniques.

获取细胞毒性和细胞对生物材料的反应

Biomaterials contact directly or indirectly with the human tissues, making it important to evaluate its cytotoxicity. This evaluation can be performed by ...