Name: impression cytology of the lid wiper area
Uploaded: 2016-08-09T15:00:00.000+00:00
Duration: 7 min 1 s
Description: Watch this Scientific Journal Video about Impression Cytology of the Lid Wiper Area at JoVE.com

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><tbody><tr><td>Alcian Blue, 1% in 3% Acetic Acid</td><td>Sigma</td><td>B8438-250ML</td><td></td></tr><tr><td>Hematoxylin, Gill 1</td><td>Sigma</td><td>GHS116-500ML</td><td></td></tr><tr><td>Papanicolaou stain, OG-6</td><td>Sigma</td><td>HT40116-500 ml</td><td></td></tr><tr><td>Papanicolaou stain, Modified EA</td><td>Sigma</td><td>HT40232-1L</td><td></td></tr><tr><td>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><td>Alcaine (0.5% proparacaine hydrochloride)</td><td>Alcon</td><td></td><td></td></tr><tr><td>Millicell Cell Culture Insert, 12&amp;nbsp;mm, hydrophilic PTFE, 0.4&amp;nbsp;&amp;micro;m</td><td>EMD Millipore</td><td>PICM01250&amp;nbsp;</td><td></td></tr><tr><td>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 ...

impression-cytology-of-the-lid-wiper-area

Research

JoVE Journal

Medicine

10.4K 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

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

科研

癌症研究

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

室管膜纤毛在小鼠脑侧脑室实时成像

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

神经科学

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

鸡颅神经嵴细胞培养物的制备及形态学分析

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

发育生物学

使用眼前节 HR-OCT 评估眼表鳞状瘤变

Anterior High-Resolution Optical Coherence Tomography in the Diagnosis and Therapeutic Monitoring of Ocular Surface Squamous Neoplasia

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. Traditionally, the diagnosis of OSSN relies on histopathological confirmation followed by a full-thickness biopsy. However, in the past two decades, the therapeutic approach to OSSN has shifted from surgical intervention to topical chemotherapy regimens in clinical settings. This shift emphasizes the need for less invasive or non-invasive methods to diagnose ocular surface pathologies. Among various imaging devices, commercially available high-resolution optical coherence tomography (HR-OCT) has emerged as a powerful tool for the characterization of OSSN. HR-OCT provides an in vivo, cross-sectional view of ocular surface lesions, offering an "optical biopsy" for OSSN with high sensitivity and specificity. It provides valuable information in differentiating intraepithelial or invasive OSSN from other benign lesions. Additionally, HR-OCT can be used to monitor the response to topical chemotherapy and to detect subclinical OSSN during follow-up visits. In this article, the scanning protocol for image acquisition is presented, and image interpretation for OSSN is outlined. This standardized, practical, and reproducible approach is recommended in clinical workflows and is expected to assist clinicians in the management of OSSN.

作者聚焦： 前部 HR-OCT 作为表征眼表鳞状瘤变的非侵入性工具

Anterior segment high-resolution optical coherence tomography (HR-OCT) is a promising non-invasive modality for the diagnosis and therapeutic evaluation of ocular surface squamous neoplasia (OSSN). Here, the system setup, scanning technique, and representative diagnostic results are presented.

前路高分辨率光学相干断层扫描在眼表鳞状瘤变诊断和治疗监测中的应用

Ocular surface squamous neoplasia (OSSN) is the most common tumor of the ocular surface, ranging from mild dysplasia to invasive squamous carcinoma. ...

工程学

A Non-invasive Way to Isolate and Phenotype Cells from the Conjunctiva

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is that they may induce traumatic lesions on the surface of the eye, which can progress to scarring, eyelid deformity, limbal stem cell deficiency and in some cases, cause great discomfort to the subject. To avoid these clinical problems, impression cytology (IC) was developed to diagnose dry eye disease and later neoplasia, atopic disease, vernal keratoconjunctivitis and keratoconjunctivitis sicca. Typically, clinicians manually cut filter papers into required shapes and apply these to the ocular surface. Here, we describe how to perform IC using a commercially available medical device. This technique is explained here followed by immunophenotyping by flow cytometry. This technique requires less manual handling and causes less injury to the ocular surface.

The exposed normal ocular surface consists of cornea and conjunctiva. Epithelial cells, goblet cells and immune cells are present in the conjunctiva. Here, a non-invasive, technique of impression cytology is described using an impression cytology device and flow cytometry to analyze immune cells in the conjunctiva.

从结膜分离和表型细胞的非侵入性方式

Traditionally, ocular surface cytology is studied with techniques such as spatula technology and brush technology. The problem with these techniques is ...

免疫与感染

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &ndash; Adaptation for the COVID-19 Pandemic

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed due to challenges with different diagnostic modalities. Ciliary videomicroscopy, using Digital High-Speed Videomicroscopy (DHSV), one of the diagnostic tools for PCD, is considered the optimal method to perform ciliary functional analysis (CFA), comprising of ciliary beat frequency (CBF) and beat pattern (CBP) analysis. However, DHSV lacks standardized, published operating procedure for processing and analyzing samples. It also uses living respiratory epithelium, a significant infection control issue during the COVID-19 pandemic. To continue providing a diagnostic service during this health crisis, the ciliary videomicroscopy protocol has been adapted to include adequate infection control measures.
Here, we describe a revised protocol for sampling and laboratory processing of ciliated respiratory samples, highlighting adaptations made to comply with COVID-19 infection control measures. Representative results of CFA from nasal brushing samples obtained from 16 healthy subjects, processed and analyzed according to this protocol, are described. We also illustrate the importance of obtaining and processing optimal quality epithelial ciliated strips, as samples not meeting quality selection criteria do now allow for CFA, potentially decreasing the diagnostic reliability and the efficiency of this technique.

Nasal Brushing Sampling and Processing Using Digital High Speed Ciliary Videomicroscopy &#8211; Adaptation for the COVID-19 Pandemic

To guarantee a successful and high-quality ciliary functional analysis for PCD diagnosis, a precise and careful method for respiratory epithelium sampling and processing is essential. To continue providing PCD diagnostic service during the COVID-19 pandemic, the ciliary videomicroscopy protocol has been updated to include appropriate infection control measures.

使用数字高速睫状视频显微镜进行鼻刷采样和处理 – 适应 COVID-19 大流行

Primary Ciliary Dyskinesia (PCD) is a genetic motile ciliopathy, leading to significant otosinopulmonary disease. PCD diagnosis is often missed or delayed ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

盖雨刮区的印象细胞学

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此视频中的章节

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

室管膜纤毛在小鼠脑侧脑室实时成像

脉络丛上皮细胞的纤毛运动的观察

鸡颅神经嵴细胞培养物的制备及形态学分析

前路高分辨率光学相干断层扫描在眼表鳞状瘤变诊断和治疗监测中的应用

前路高分辨率光学相干断层扫描在眼表鳞状瘤变诊断和治疗监测中的应用

前路高分辨率光学相干断层扫描在眼表鳞状瘤变诊断和治疗监测中的应用

从结膜分离和表型细胞的非侵入性方式

使用数字高速睫状视频显微镜进行鼻刷采样和处理 – 适应 COVID-19 大流行

视网膜平片准备成像和免疫组化优化方案