Name: scanning electron microscopy of macerated tissue to visualize the extracellular matrix
Uploaded: 2016-06-14T15:00:00.000+00:00
Duration: 10 min 21 s
Description: Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

This study was funded by grants from the National Institutes of Health (NIH), Heart, Lung, and Blood Institute (NIHLB): K01-HL-121045, K08-HL-094703, 5T32HL007411-35, P20 HL101425, U01 HL100398. 
Imaging and tissue processing (after NaOH maceration) were performed through the use of the Vanderbilt University Medical Center (VUMC) Cell Imaging Shared Resource (CISR) (supported by NIH grants CA68485, DK20593, DK58404, DK59637 and EY08126). We are especially grateful to the VUMC CISR core directors (Dr. Sam Wells and Dr. W. Gray (Jay) Jerome) for valuable technical advice and also for providing core space and resources for the purposes of filming the technique highlighted in this paper. 
We would like to extend our deepest appreciation to Dr. Yan Ru Su and Ms. Kelsey Tomasek in the Cardiology Core Lab for Translational and Clinical Research at Vanderbilt University for providing technical expertise and for collecting human tissue samples used in this study.

伦理声明:动物处理的协议是由范德比尔特机构动物护理和使用委员会（IACUC批准，根据AAALAC -国际标准协议数M / 10/117（猪）和M / 10/219（小鼠）和进行。开户人的心脏组织的使用是经范德比尔特大学医学中心IRB（协议号为100887）。 1.样品采集和存储<ol><li>使在0.1M磷酸盐缓冲液（PB）的溶液新鲜的4％戊二醛。 注意:戊二醛是有毒的，戴手套和工作在通风橱。 <ol><li>使用21.8克Na 2 HPO 4和6.4克的NaH 2 PO 4。 量子SATIS（QS）的PB，pH为7.4的0.2M的原液达1,000ml卫生署2 O. </li><li>加入400微升50％戊二醛的2.5毫升PB原液和2.1毫升卫生署2 O. </li></ol></li><li>沉浸在一个块（不大于2 C米2）的组织的成4％的戊二醛溶液中。注意:小片也可以使用，但应具有足够的尺寸（不小于5 平方毫米），以容易地通过眼睛，以便于以后的协议的步骤可视化的。 </li><li>在室温下孵育1小时，然后在4℃下无限期地存储。 </li></ol> 2.心脏组织的脱细胞<ol><li>使用10克NaOH粒料/100毫升卫生署2 O.新鲜的10％NaOH水溶液 注意:NaOH溶液具有腐蚀性，可引起碱烧伤，戴手套。 </li><li>冲洗组织在卫生署2 O. </li><li>在室温下6孵育在10％NaOH溶液 - 10天（直至从红褐色组织变为灰白色或白色）。 </li><li>在冲洗卫生署2 O，直到组织变得透明。 </li><li>浸泡在1％的单宁酸的组织，在室温下4小时。使用每4毫升卫生署2 O.1毫升5％原液 注意:单宁酸强烈的刺激性，戴手套。 </li><li>在冲洗卫生署2 O过夜。 </li></ol> 3. Osmication和心脏组织脱水（在通风橱安全） <ol><li>使用21.4克二甲胂酸钠10.0g二氯化钙和450毫升卫生署2 O的二甲胂酸钠缓冲液中的0.2M的原液混合，然后根据需要将pH调节至7.4 QS添加盐酸。到500毫升的dh 2 O 注意:二甲胂酸钠和盐酸是有毒的，戴手套，工作在通风橱中。 </li><li>使在通风橱安全四氧化锇的2％水溶液原液通过在50毫升的dh 2 O1克四氧化锇晶体溶解 注意:四氧化锇是一个重度吸入性危害;粘膜或眼球蒸汽固定是可能的，因此，只有在带着手套通风柜处理。推荐使用防溅板。 </li><li>在冲洗0.1M的二甲胂酸钠缓冲组织（1混合原液:1与卫生署<suB&gt; 2 O）对转子5分钟（或轻柔搅拌）。 </li><li>重复先前的步骤两次，总共三个缓冲器漂洗。 </li><li>浸泡在1％的四氧化锇组织在0.1M二甲胂酸钠缓冲液（混合二甲胂库存钠和库存的四氧化锇1:1）上旋转1小时。 </li><li>冲洗组织在0.1M二甲胂酸钠缓冲液3次，每次在旋转器5分钟。 </li><li>使用的乙醇浓度增加（30％，50％，75％，85％，95％，最后100％）对转子每15分钟冲洗组织。 </li></ol> 4.截面表面准备SEM <ol><li>在100％乙醇转移组织以一个浅培养皿还含有100％的乙醇。 </li><li>容纳两个非常尖锐的刀片，使得双方的单位都与彼此和切削刃交叉接触，以形成检体上方的等边三角形的两个方面。要做到这一点，用左手放置一个刀片对样品的最右侧，切向左。在同一时间，用右手放置在第二刀片上最左边的向右样品和切片。因此，刀片会互相从相反的方向滑动，以一个单一的顺利晋级。 </li><li>滑动平刃双方对对方做出最小失真或撕裂力的标本非常干净的切割，最好露出尽可能大的表面积尽可能不破坏样品。 </li><li>重复每个标本暴露清洁的横截面表面在扫描电镜检查。 </li></ol>心脏组织5.临界点干燥（CPD） <ol><li>用刮刀或镊子组织转移到临界点干燥器（CPD）的样品架，以确保组织在任何时候都保持在100％的乙醇。确保该支架被浸入在乙醇中，该传送与暴露于空气中不超过几秒钟的组织来实现。 </li><li>每个操作CPD我们呃手册完成3吹扫和填充循环用液体二氧化碳来代替乙醇。 </li><li>操作每个用户的手册CPD实现样品的临界点干燥，并将它们与二氧化碳的控制排气返回到大气压力。 </li></ol> 6.安装心脏组织样本进行扫描电镜<ol><li>制备的SEM样品存根为每个试样，通过粘附碳粘合剂标签，所述铝存根的顶表面。 </li><li>用立体显微镜的帮助下，小心地附着试样到与感兴趣的横截面表面的粘合剂标签朝上（从标签中，可见的距离），并与样品存根表面的平面尽可能接近平行。不要探头或触摸的感兴趣的表面。 </li><li>打破木制涂药棒，实现了锥形刷，非常适合涂料应用。在基座和试样的两侧施加银或碳涂料，以提高粘附到存根。 </li><li>延伸的非常薄的银或碳的线画到感兴趣的表面的边缘，以提供从感兴趣的地面表面的电荷的路径。 </li><li>应用银或碳涂料的2或3小的dAb围绕碳片的周边，以提供从碳片表面到金属短截线的导电路径，从而接地。 </li><li>允许导电性涂料干燥两小时。 </li><li>操作每个用户的手册溅射镀膜机施加相对重的涂层（30目标范围 - 40纳米）金 - 钯合金或金。泵样品室约0.1毫巴;设置定时器为40秒。打开设置阀8:00位置（中等氩气流量）。按Start开始溅射镀膜在30毫安。紫色辉光放电应在试样腔室是可见的。 </li></ol> 7.心脏组织样本的扫描电镜检查<ol><li>执行在相对低的加速电压的扫描电子显微镜，以减少成像p与样品中电荷耗散不良有关roblems（充电）。建议初始成像条件是:5千伏加速电压10毫米的工作距离。 </li><li>用一个有经验的操作者的协助下，雇用增加的工作距离，以在需要用于在z方向相当长延伸的多个焦平面纤维，或纤维的焦点成像条件延伸景深。 <ol><li>对于这里使用的显微镜（见材料表 ），在用户界面访问在右上角导航选项卡。 </li><li>从舞台菜单进入坐标标签。以增加的工作距离，以毫米为在Z输入一个较大的值的坐标，然后就转到点击到标签到样品台移动到输入工作距离。 </li></ol></li><li>用一个有经验的操作者的协助下，采用试样倾斜和旋转到兴趣正交的表面定位到电子束。的10至30 DEGR附加倾斜EES从该位置可以改进基体结构的观察和记录。 <ol><li>对于这里使用的显微镜（见材料表 ），在用户界面中，单击并按住鼠标右键，然后向左或向右滑动关注感兴趣的准备好的表面平面的外围附近的样本，并指出聚焦工作距离。 </li><li>与手动用户界面摇杆导航到附近的表面和重复焦点的相对边缘移动。如果工作距离不近似等于所述第一位置，倾斜检体来实现在使用Stage菜单的坐标标签（7.2），并进入在T的倾斜坐标值两个位置近似一致。 </li><li>旋转样品九十度（输入值成R坐标场）和直至所有的位置都集中在大致相同的工作距离重复上述过程。 注意:可变压力SEM可以用于改善电荷耗散如果可供使用的话。高真空是用于扫描电子显微镜的标准操作模式。 </li></ol></li></ol>

The authors have nothing to disclose.

横截面表面处理是协议中最关键的一步。为了保持精细结构，脱水标本必须在任何时候都保持在100％的乙醇，直到引入到临界点干燥工序。因此样品的切片，以实现EM检查表面必须同时标本在乙醇浸没在浅盘来完成。它也是至关重要的暴露表面不触及或后续处理期间探测。任何重大修改，预计在这一技术到其他组织类型相似矩阵的观测应用程序，不过协议的SEM部分可能需要基本的电子显微镜故障?...

Fibrosis disrupts the normal myocardial collagen network, which is critical for normal mechanistic functions of cardiomyocytes 1,2 as well as for inter-cellular communication, intracellular signaling, and cell survival 3. The development of fibrosis is a major determinant of cardiac function, and fibrotic remodeling of the cardiac interstitium arising from a variety of etiologies leads to increased left ventricular stiffness and diastolic dysfunction 4. Myocardial fibrosis may also lead to arrhythmias, and whether the progression of fibrotic remodeling is a general or local phenomenon, it is highly associated with a poor prognosis in patients with ischemic and non-ischemic cardiomyopathy 5. Likewise, the absence of myocardial fibrosis is a strong predictor of ventricular functional recovery and long-term survival 6. 
The hallmark of fibrosis is the deposition of excess collagen, which has the tensile strength of steel 7 and can adversely affect cardiomyocyte function on multiple levels. Mechanical forces resulting from an excessively collagenous matrix can lead to cardiomyocyte atrophy 8,9, passive tissue stiffness 10, tonic contraction-induced myocardial stiffness 11-13, and reduced delivery of oxygen to the remaining population of cardiomyocytes. Gap junction coupling of cardiomyocytes and myoFbs can also compromise the heart's electrical characteristics, creating a greater risk for the development of arrhythmias 14-16. Perivascular fibrosis alters vasomotor reactivity of intramural coronary arteries and arterioles 17 and contributes to luminal narrowing that reduces the supply of oxygen and thus the survival of cardiomyocytes 17-22. Pathogenic fibrotic and electrical remodeling, emanating from an initial site of ischemic injury or energy imbalance, inevitably progresses to heart failure. 
Cardiomyocyte necrosis initiates the fibrotic response, and subsequent adverse fibrotic remodeling can occur irrespective of etiology. Finding a way to control cardiac fibrosis would be clinically beneficial for the treatment of ischemic and idiopathic cardiomyopathies, hypertensive heart disease, hypertrophic cardiomyopathy, valvular heart disease and dystrophinopathies 23-42. Regardless of how the fibrotic disease process begins, soluble, profibrotic factors can cross the interstitial space and provoke activation of interstitial and adventitial fibroblasts at sites remote to the initial fibrotic scar, creating a cascade effect that ultimately leads to heart failure. The optimum scenario would be to exploit the fibrillogenic process using a targeted therapeutic that can be applied during the compensative hypertrophic stage of cardiomyopathy before it progresses to systolic pump failure, diastolic heart failure, or other end-stage outcomes. The ultimate goal would be to reverse fibrosis so that dead cardiomyocytes can be replaced and heart function restored completely. 
The importance of the matrix is widely understood, yet methods to study the matrix are limited mainly to quantitative measurements of major structural components, particularly collagen, and relative levels of different matrix and matricellular proteins. This protocol highlights a rarely used technique that is useful for assessing qualitative differences in the cardiac matrix. This technique has been recently used to compare and contrast fundamental differences in heart matrices from different etiologies of heart disease (in human explants), to examine hearts from post-infarcted pigs treated with the glial growth factor (GGF) isoform of neuregulin-1&#946;, relative to untreated animals 43, and to probe for differences in the matrices of cardiac tissues from mdx mice (a commonly used animal model of Duchenne Muscular Dystrophy) at different ages and compared to wild-type controls. This technique was first introduced by Drs. Caulfield and Borg in 1979 44, but few studies have since employed this powerful technique 45-47, re-introduced here with only slight modification. This methodology is a valuable research tool, because it provides qualitative information about extracellular matrix ultrastructure that might otherwise be overlooked when simply measuring matrix component content and/or level of fibrosis.

<table><tbody><tr><td>Calcium Chloride</td><td>Electron Microscopy Sciences</td><td>12340</td><td>100 g</td></tr><tr><td>Carbon Adhesive</td><td>Electron Microscopy Sciences</td><td>12664</td><td>30 g</td></tr><tr><td>Carbon Adhesive Tabs</td><td>Electron Microscopy Sciences</td><td>77825</td><td>order to fit stubs</td></tr><tr><td>Double Edge Razor Blades Stainless Steel</td><td>Ted Pella, Inc</td><td>121-6</td><td>250/pkg</td></tr><tr><td>Ethanol</td><td>Electron Microscopy Sciences</td><td>15055</td><td>450 ml</td></tr><tr><td>Gluteraldehyde, 50% Solution</td><td>Electron Microscopy Sciences</td><td>16310</td><td>EM grade, distillation purified</td></tr><tr><td>Hydrochloric Acid</td><td>Electron Microscopy Sciences</td><td>16760 or 16770</td><td>100 ml</td></tr><tr><td>Monosodium Phosphate NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9251-250G</td><td>250 g</td></tr><tr><td>Osmium Tetroxide</td><td>Electron Microscopy Sciences</td><td>19100</td><td>1 g</td></tr><tr><td>Silver Conductive Adhesive</td><td>Electron Microscopy Sciences</td><td>12686-15</td><td>15 g</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>S8045-1KG</td><td>1 kg</td></tr><tr><td>Sodium Phosphate Dibasic (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S3264-500G</td><td>500 g</td></tr><tr><td>Tannic Acid, 5% Aqueous&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>21702-5</td><td>500 ml</td></tr><tr><td>Trihydrate Sodium Cacodylate</td><td>Electron Microscopy Sciences</td><td>12300</td><td>100 g</td></tr><tr><td>Gold-palladium Alloy or Gold</td><td>Refining Systems, Inc.&amp;nbsp;</td><td>varies</td><td>specific to the sputter coater make and model</td></tr><tr><td>Critical Point Dryer</td><td>Electron Microscopy Sciences</td><td>850</td><td></td></tr><tr><td>Plain Wooden Applicators</td><td>Fisher Scientific</td><td>23-400-102</td><td></td></tr><tr><td>Quanta 250 Environmental SEM</td><td>FEI</td><td>Q250 SEM</td><td></td></tr><tr><td>Sputter Coater</td><td>Cressington Scientific Instruments Ltd.</td><td>Model 108</td><td></td></tr><tr><td>Alluminum SEM Sample Stubs</td><td>Electron Microscopy Sciences</td><td>75220-12</td><td>specific to the miscroscope</td></tr></tbody></table>

scanning electron microscopy of macerated tissue to visualize the extracellular matrix

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix biology, there are still major gaps related to how the matrix is formed, how physiological and pathological remodeling differ, and most importantly how matrix dynamics might be manipulated to promote healing and inhibit fibrosis. There is currently no treatment option for controlling, preventing, or reversing cardiac fibrosis. Part of the reason is likely the sheer complexity of cardiac scar formation, such as occurs after myocardial infarction to immediately replace dead or dying cardiomyocytes. The extracellular matrix itself participates in remodeling by activating resident cells and also by helping to guide infiltrating cells to the defunct lesion. The matrix is also a storage locker of sorts for matricellular proteins that are crucial to normal matrix turnover, as well as fibrotic signaling. The matrix has additionally been demonstrated to play an electromechanical role in cardiac tissue. Most techniques for assessing fibrosis are not qualitative in nature, but rather provide quantitative results that are useful for comparing two groups but that do not provide information related to the underlying matrix structure. Highlighted here is a technique for visualizing cardiac matrix ultrastructure. Scanning electron microscopy of decellularized heart tissue reveals striking differences in structure that might otherwise be missed using traditional quantitative research methods.

突出显示的技术应用于心脏组织从一个未使用的人体心脏移植供体（ 图1），从移植，从野生型和营养不良的小鼠（ 图3）的心外植组织和从猪心肌梗死后心脏的样品中心脏损伤模型（ 图2）。 如图1所示，人的心脏基质的横截面中观察时显示一个蜂巢状图案的交联蛋白质的一种复杂的编织。每个&#39;蜂窝&#39;结构是约40微米...

Watch this Scientific Journal Video about Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix at JoVE.com

Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

扫描浸软组织的电子显微镜可视化的细胞外基质

Fibrosis is a component of all forms of heart disease regardless of etiology, and while much progress has been made in the field of cardiac matrix ...

scanning-electron-microscopy-macerated-tissue-to-visualize

Research

JoVE Journal

Biology

9.9K 次观看.  Vanderbilt University Medical Center. Shown here is a method for visualizing extracellular matrix ultrastructure in decellularized cardiac tissues.

视频: Scanning Electron Microscopy of Macerated Tissue to Visualize the Extracellular Matrix

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

科研

生物化学

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

观察和量化纤维细胞介导的纤维素凝胶压实

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

生物工程

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Visualizing Macrophage Extracellular Traps Using Confocal Microscopy

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal microscopy methods to visualize macrophage extracellular traps (METs). These extracellular traps are defined by the presence of extracellular chromatin with co-expression of other components such as granule proteases, citrullinated histones, and peptidyl arginase deiminase (PAD). The expression of METs is generally measured after exposure to a stimulus and compared to un-stimulated samples. Samples are also included for background and isotype control. Cells are analyzed using well-defined image analysis software. Confocal microscopy may be used to define the presence of METs both in vitro and in vivo in lung tissue.

Macrophage extracellular traps are a newly described entity. This article will concentrate on confocal microscopy methods and how they are visualized in vitro and in vivo from lung samples.

利用共焦显微镜可视化巨噬细胞胞外陷阱

A primary method used to define the presence of neutrophil extracellular traps (NETs) is confocal microscopy. We have modified established confocal ...

免疫与感染

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

生物学

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 ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

<meta charset="utf8"></head><body>穿刺伤口止血和蒙太奇电子显微镜

Puncture Wound Hemostasis and Preparation of Samples for Montaged Wide-Area Electron Microscopy Analysis

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds from time to time. In hemostasis, self-limiting platelet aggregation leads to the formation of a structured thrombus in which bleeding cessation comes from capping the hole from the outside. Detailed characterization of this structure could lead to distinctions between hemostasis and thrombosis, a case of excessive platelet aggregation leading to occlusive clotting. An imaging-based approach to puncture wound thrombus structure is presented here that draws upon the ability of thin-section electron microscopy to visualize the interior of hemostatic thrombi. The most basic step in any imaging-based experimental protocol is good sample preparation. The protocol provides detailed procedures for preparing puncture wounds and platelet-rich thrombi in mice for subsequent electron microscopy. A detailed procedure is given for in situ fixation of the forming puncture wound thrombus and its subsequent processing for staining and embedding for electron microscopy. Electron microscopy is presented as the end imaging technique because of its ability, when combined with sequential sectioning, to visualize the details of the thrombus interior at high resolution. As an imaging method, electron microscopy gives unbiased sampling and an experimental output that scales from nanometer to millimeters in 2 or 3 dimensions. Appropriate freeware electron microscopy software is cited that will support wide-area electron microscopy in which hundreds of frames can be blended to give nanometer-scale imaging of entire puncture wound thrombi cross-sections. Hence, any subregion of the image file can be placed easily into the context of the full cross-section.

<meta charset="utf8"></head><body>作者聚焦:通过结构血液学的进步揭示血小板动力学

A puncture wound procedure for hemostatic thrombus formation is presented here. The formed thrombi are large and are hundreds of microns in diameter. Hence, volume imaging approaches are appropriate. We suggest montaged wide-area transmission electron microscopy as a high-resolution approach available to many and detail a preparative protocol.

穿刺伤口止血和蒙太奇广域电子显微镜分析样品的制备

Hemostasis, the process of normal physiological control of vascular damage, is fundamental to human life. We all suffer minor cuts and puncture wounds ...

扫描浸软组织的电子显微镜可视化的细胞外基质

摘要

探索更多视频

此视频中的章节

细胞外基质的Glycoproteomics:使用质谱联用完整糖肽分析的方法

观察和量化纤维细胞介导的纤维素凝胶压实

与落射荧光组织基质成分和双光子显微镜的长期活体免疫荧光成像

提高2D和3D皮肤

利用共焦显微镜可视化巨噬细胞胞外陷阱

定量测量的侵袭伪足介导的细胞外基质蛋白水解单和多细胞的情况下

从富集组织及消化细胞外基质蛋白的成肽质谱分析

一种快速，为隔离，功能可扩展的研究方法，以及细胞衍生的细胞外基质的分析

穿刺伤口止血和蒙太奇广域电子显微镜分析样品的制备

穿刺伤口止血和蒙太奇广域电子显微镜分析样品的制备