Name: a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy
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Description: Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

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注:所有涉及使用动物的程序进行，根据当地的法规。我们的工作是经悉尼当地卫生区动物福利委员会。允许程序项目许可文档中描述，并按照指引，以确保动物在任何时候都幸福。确保遵守有关的地方进行工作的国家动物实验的立法。 为准备EM固色剂1.协议<ol><li> 100ml中的固定剂的，通过加入2多聚甲醛粉末至25ml在一个锥形瓶中蒸馏水，加​​热至65ºC，用磁力搅拌器不断搅拌制备的多聚甲醛。 注:戊二醛，多聚甲醛和二甲胂酸钠缓冲都是有害物质，必须始终在通风柜处理。 </li><li>热，在65ºC为2分钟，然后允许冷却。如果需要的话，加氢氧化钠溶液dropwISE边搅拌至完全明确的解决方案。 注:氢氧化钠是危险的，小心处理。 </li><li>加入10毫升EM级库存戊二醛，将50ml的0.2M二甲胂酸钠缓冲液和2克蔗糖和搅拌用磁力搅拌器。添加1.0M的氯化钙2 2毫升</li><li>调节至pH 7.4之前使溶液至100毫升蒸馏水。最终固定剂中含有2.5％戊二醛，2％多聚甲醛，2毫摩尔氯化钙 ，2％蔗糖的0.1M二甲胂酸盐缓冲液中。确保总渗透压为440 mOsmol /升。使用内准备24小时。 </li></ol>肝2.灌注固定<ol><li>温馨灌注缓冲和定于35 - 37ºC。放血的缓冲液和固定液的正确的温度有助于确保组织的完整性。 </li><li>麻醉的动物用单次腹膜内注射50毫克/千克氯胺酮和5mg / kg的甲苯噻嗪。 </li><li>一旦动物是下深度麻醉，用后肢撤退和尾掐评估，A Y切口，钝性结束剪刀在动物的腹部，露出肝脏和门静脉。 注:确保设备清洁，避免标本微生物和一般的碎片，这将很容易看到SEM污染。 </li><li>扎两个非常松散的缝线门静脉周围1近侧至肝脏，在其他更远侧到肝脏。 </li><li>导管插入门静脉与适当大小的IV套管（18克的老鼠，22克的老鼠）.Tighten缝线固定套管。 </li><li>开始灌注用磷酸盐缓冲盐水加热到37ºC，并在10厘米的H 2 O的压力。 5至20毫升的PBS通常需要抽血肝脏。 注意:不要让气泡进入通过连接到他们的文物引起继发栓塞和压力套管管肝脏;损害肝细胞，SInusoids和LSEC窗孔发生，如果灌注压力过高。 </li><li>切断的腹部和胸部腔静脉，以允许缓冲从肝脏自由退出。这可以防止高背压损坏LSECs。 </li><li>一旦肝脏是免费的血液，用EM固定液更换PBS和灌注约5分钟，直到肝脏硬化，很苍白。 </li><li>停止灌注和削减固定肝脏中转化为1 - 2 mm 3的街区用锋利的手术刀。 </li><li>后修复组织EM固定液24 - 72小时，在4ºC。 注:在一般情况下，underfixation导致比overfixation更多的文物。 </li><li>更改固定液以下后修复期为存储0.1M的二甲胂酸钠缓冲液，4ºC。 注:存储在这种方式标本将被保存为一个显著一段时间（最长12个月），但及时的辅助固定和审查建议以获得最佳效果。 </li></ol> 3.针Perfusion 注:针固定是灌注固定的一个变种，涉及到注入固定液直接进入肝脏活检组织。这样做的目的是为固定剂可以通过血管，以抽血它们和公开所有的组织块以固定剂冲洗。必须小心，以保持注射压力低，以避免气压伤18,19。 <ol><li>取出一小片来自动物或受肝脏和冲洗的生理盐水，以清除血液和碎片。 </li><li>绘制生理盐水注射进装有细针头（通常胰岛素注射器）。 </li><li>注入生理盐水非常缓慢进入肝脏的血，直到被刷新出来的组织。多次注射可能是必需的。 </li><li>绘制对EM固定液到装配有细规针（通常是胰岛素注射器）另一个注射器。 </li><li>注入固定液非常缓慢进入肝脏，直到它变得坚硬和棕褐色。多次喷射之间可以需要。 </li><li>用锋利的手术刀2毫米3个街区-切肝固定为1。在孵育固定液EM为24 - 72小时4℃。开始二次固定前需清洗的0.1M的二甲胂酸钠缓冲液的3倍。 </li></ol> 4.准备扫描电子显微镜<ol><li>洗涤样品在0.1M二甲胂酸钠缓冲液3次。要修复脂类，交固定在0.1M二甲胂酸钠缓冲液将样品在2％四氧化锇进行2小时。 注:完全清洗是非常重要的，因为戊二醛和四氧化锇交叉反应，生成SEM文物。 </li><li>冲洗样品中增加的乙醇浓度开始脱水。第一冲洗在50％乙醇5分钟;然后3次，每次5分钟，用70％的乙醇;冲洗3次，每次5分钟，用90％的乙醇;冲洗2次，每次5分钟，在100％乙醇;最后漂洗2次，在100％乙醇（分子筛）10分钟。 </li><li>从样本中删除所有乙醇<html>和六甲基二带（HMDS）代替通风柜，离开10分钟。从组织样品中除去HMDS，并允许蒸发。 注:HMDS是吸湿性强冷的时候，因此允许使用的最终脱水行程之前达到室温。 </li><li>可选，立即装入样品或将它们放入干燥器中，直到准备安装的扫描电镜。 </li></ol> 5.安装注:正确的样品安装将最大限度的提供SEM分析下明确界定血窦的数量。 <ol><li>标记金属SEM存根的基极与应用双面导电碳胶带的存根的顶部。 </li><li>使用解剖显微镜来识别与用于随后的SEM最好血窦表面可视化的样品。向上放置该表面上的截线的碳胶带表面。 </li><li>坚持标本坚决存根，样品现在已经准备好了溅射镀膜。 </li></ol>ove_title"&gt; 6。涂料注:涂层在溅射镀膜机的理由有导电金属（金或铂）的致密的膜试样的试样和从电子束损伤保护它。如果涂层是感兴趣太厚结构可以被遮蔽。 <ol><li>地方存根成的自动微溅射镀膜机的真空室，45秒设置模式为旋转和自动溅射涂覆。使用铂涂层更精细的细节，但是金也是合适的。 </li><li>涂抹一层薄薄的碳涂料以所安装的肝标本的外表面，小心不要涂覆正弦曲面。 </li><li>一旦标本都涂有铂和碳​​油漆已干，他们已经准备好观察的SEM。样品可以在干燥器中储存。 </li></ol> 7.使用扫描电子显微镜<ol><li>地方标本进入扫描电子显微镜，并返回显微镜至真空。&lt;/ LI&gt; <li>使用的样本可视化标准显微镜操作程序通过在开始低倍率，逐渐增加的放大倍率。 </li><li>确保显微镜为工作距离，光点尺寸和散光适当调整。 </li><li>检查固定的完整性。如果LSEC是从缺口较自由，并包含窗孔测量30 - 250 nm的直径，这将通常表明试样制备已经成功。 </li><li>如果正弦充满空隙或缺乏穿孔的试样准备尚未成功或有显著病理。在这种情况下，片具有非常优良的剃刀刀片的样品，并且切割表面重新涂复并成功地固定正弦分析。 </li><li>大约在15,000-20,000×倍率选择平坦的表面LSEC是免费的碎片具有良好的固定和其中窗孔清晰可见（通常至少含有10的窗孔）。 </li><li>保存至少10血窦图像每肝，来自各块的不同区域，使用每肝的至少两个块。采样10的显微照片以15,000 - 每只动物20000×放大倍数通常是足够的窗孔的量化是代表整个肝脏的。 </li></ol> 8.分析注意:可从美国国立卫生研究院免费下载的ImageJ软件来定量测定开窗直径和频率（ <a href="http://imagej.nih.gov/ij">www.imagej.nih.gov/ij</a> ）。 <ol><li>使用嵌入在图像（ 屏幕快照1）的比例尺打开图像J和设置比例。 </li><li>使用多边形工具中的ImageJ，跟踪绕LSEC的整个平面区域包括有孔和defenestrated领域。不要只跟踪筛板，因为这会错误地夸大孔隙度。排除任何大的碎片是在细胞表面上，可能是obscuriNG窗孔。 </li><li>为了测量直径开窗，通过选择行工具跟踪通过每个开窗的最长直径线，然后按"M"来衡量的路线，和"d"（画）永久画上的图片就行了。这条线被定义为开窗直径。该生产线的长度现在可在结果框中的ImageJ（ 屏幕打出2）。 </li><li>测量这些都是LSEC细胞质超过250纳米的较大的孔，以及窗孔所有间隙。间隙往往假象反射灌注或定影不良，但是间隙也可以作为疾病和毒性的结果。对于缺口的数据将被排除在开窗参数计算分析后。 </li><li>差距是不是圆形的区域应该通过跟踪周围的空白，并计算其在ImageJ的面积计算。 </li><li>从ImageJ的结果框中的数据粘贴到Excel电子表格的furthe- [R分析。 </li></ol> 9.计算<ol><li>平均直径开窗平均=所有开窗直径（不包括空白，其中差距≥250纳米）。 </li><li>开窗面积=πR2，其中r，半径，是从各个窗孔直径相关（r = D / 2），不包括间隙算出。 </li><li>孔隙率（％）=（Σ（πR2）/分析总面积- Σ（间隙的面积=））（微米2））×100。 </li><li>开窗频率=开窗总数/（总面积分析-Σ（空白区域）（微米2）。 </li></ol> 10.介绍开窗数据注:只要有可能，出版物，包括开窗数据的量化应包括下列资料<ol><li>开窗直径，以声明证实什么用的地方直径边界定义开窗（通常间50 - 250纳米），开窗频率（数个/μm2）和孔隙率（％）。 </li><li>声明确认是否间隙已被包括在分析中，开窗直径和肝脏，块，图像和窗孔的数目的频度分布曲线应包括在分析中。 </li></ol>

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A. 108 (7), 2945-2950 (2011).</li><li>Zhang, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OxLDL+induced+injury+and+defenestration+of+HLSECs+via+LOX-1.">OxLDL induced injury and defenestration of HLSECs via LOX-1.</a> J Mol Endocrinol. , (2014).</li><li>May, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+model+for+conditional+induction+and+rescue+of+portal+hypertension+reveals+a+role+of+VEGF-mediated+regulation+of+sinusoidal+fenestrations.">A transgenic model for conditional induction and rescue of portal hypertension reveals a role of VEGF-mediated regulation of sinusoidal fenestrations.</a> PLoS One. 6 (7), e21478 (2011).</li><li>Funyu, J., Mochida, S., Inao, M., Matsui, A., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGF+can+act+as+vascular+permeability+factor+in+the+hepatic+sinusoids+through+upregulation+of+porosity+of+endothelial+cells.">VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells.</a> Biochem Biophys Res Commun. 280 (2), 481-485 (2001).</li><li>Venkatraman, L., Tucker-Kellogg, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CD47-binding+peptide+of+thrombospondin-1+induces+defenestration+of+liver+sinusoidal+endothelial+cells.">The CD47-binding peptide of thrombospondin-1 induces defenestration of liver sinusoidal endothelial cells.</a> Liver Int. 33 (9), 1386-1397 (2013).</li><li>Wack, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinusoidal+ultrastructure+evaluated+during+the+revascularization+of+regenerating+rat+liver.">Sinusoidal ultrastructure evaluated during the revascularization of regenerating rat liver.</a> Hepatology. 33 (9), 363-378 (2001).</li><li>Morsiani, E., Mazzoni, M., Aleotti, A., Gorini, P., Ricci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+sinusoidal+wall+permeability+and+liver+fatty+change+after+two-thirds+hepatectomy:+An+ultrastructural+study+in+the+rat.">Increased sinusoidal wall permeability and liver fatty change after two-thirds hepatectomy: An ultrastructural study in the rat.</a> Hepatology. 21 (2), 539-544 (1995).</li><li>Straub, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arsenic-stimulated+liver+sinusoidal+capillarization+in+mice+requires+NADPH+oxidase&#8211;generated+superoxide.">Arsenic-stimulated liver sinusoidal capillarization in mice requires NADPH oxidase&#8211;generated superoxide.</a> J Clin Invest. 118 (12), 3980-3989 (2008).</li><li>Barber&#225;-Guillem, E., Arrue, J. M., Ballesteros, J., Vidal-Vanaclocha, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+changes+in+endothelial+cells+of+developing+rat+liver+in+the+transition+from+fetal+to+postnatal+life.">Structural changes in endothelial cells of developing rat liver in the transition from fetal to postnatal life.</a> JJ Ultrastruct Mol Struct Res. 97 (1-3), 197-206 (1986).</li><li>Jamieson, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caloric+restriction+reduces+age-related+pseudocapillarization+of+the+hepatic+sinusoid.">Caloric restriction reduces age-related pseudocapillarization of the hepatic sinusoid.</a> Exp Gerontol. 42 (4), 374-378 (2007).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperlipidemia+and+surfactants:+the+liver+sieve+is+a+link.">Hyperlipidemia and surfactants: the liver sieve is a link.</a> Atherosclerosis. 189 (2), 273-281 (2006).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+oxidative+stress+on+the+liver+sieve.">The effects of oxidative stress on the liver sieve.</a> J Hepatol. 41 (3), 370-376 (2004).</li><li>Cogger, V. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+structured+illumination+microscopy+of+liver+sinusoidal+endothelial+cell+fenestrations.">Three-dimensional structured illumination microscopy of liver sinusoidal endothelial cell fenestrations.</a> J Struct Biol. 171 (3), 382-388 (2010).</li><li>Braet, F., de Zanger, R., Seynaeve, C., Baekeland, M., Wisse, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+atomic+force+microscopy+study+on+living+skin+fibroblasts+and+liver+endothelial+cells.">A comparative atomic force microscopy study on living skin fibroblasts and liver endothelial cells.</a> J Electron Microsc. 50 (4), 283-290 (2001).</li><li>Monkemoller, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+fenestrations+in+liver+sinusoidal+endothelial+cells+by+optical+localization+microscopy.">Imaging fenestrations in liver sinusoidal endothelial cells by optical localization microscopy.</a> Phys Chem Chem Phys. 16 (24), 12576-12581 (2014).</li></ol>

The authors have nothing to disclose.

精确地和可重复地测量所述肝窦内皮的状态的能力是在理解这些高度特化的细胞的生物学的一个重要步骤。如结构照明显微镜32，原子力显微镜33和d- STORM（直接随机光学Reconstrucion显微镜）34更新的技术将赋予重要的信息对这些细胞在体外的形态，但SEM仍然是主要的方法来可视化和在测量它们的结构原位 。 最关键和技术挑战性的一?...

肝窦内皮细胞（LSECs）是高度分化的内皮细胞排列在肝窦的壁。 LSECs均穿有穿孔是非diaphragmed，跨细胞孔径为50-250纳米。高达LSECs的表面的20％被覆盖穿孔，这通常是在几十组到几百称为筛板1-3（ 图1）。开窗使血浆和血液和肝细胞之间nanosubstrates的转移，创造一个高效的超滤系统。窗孔是动态的结构 - 无论它们的尺寸和/或数量可以响应于各种生理状态，药物和疾病而改变。例如，穿孔较大在禁食比在进食状态4; 2-二- iodoamphetamine增加开窗号; 5,6-和尺寸的减小和穿孔的数目每细胞发生衰老和许多疾病状态7-13 </sup&gt;。窗孔的尺寸和数量的精确测量是理解如何LSEC形态是由各种疾病，中毒性和生理状态的影响重要;它们对肝功能的影响;和用于显影开窗调制治疗干预1。 窗孔的研究是困难的。窗孔的直径在于传统光学显微镜的分辨率下面，所以无论是在完整的肝组织或培养LSECs以前只观察用电子显微镜已经成为可能。扫描电子显微镜（SEM）已最经常被用于研究开窗尺寸，频率和孔隙率（LSEC膜是受窗孔穿孔的百分比），因为SEM允许观测大面积的数千内皮表面和测量，如果不是几万窗孔的。尽管它的效用，这是从报告的结果的SEM为基础的研究为LSEC参数如开窗大小，数量，频率和孔隙率有很大的不同在文献中（ 表1）。 开窗和筛板是脆弱的结构，其合同，打破，扩张或标本制备过程中凝聚，从而精心加工是需要保持其完整性。升高灌注压14;固定液和缓冲区15的不正确的渗透压;固定不充分或固定时间;和后固定脱水和干燥的速度是处理用于SEM的所有领域，可能产生伪影，与保存超微结构的干扰（ 图2）。窗孔的损失（&#39;抛出窗外&#39;）和开窗收缩可发生定影不良的结果，从而降低开窗直径和孔的孔隙率。方法，提高试样的保存用于SEM分析先前已经描述15-17和将要讨论的这里，关于如何提高标本保存额外的提示。检体保存的主要目标是从血窦除去血液，使得LSEC的表面可以可视化，并避免从任一高压或延迟固定的LSEC损坏。固定剂经门静脉全肝灌注是肝脏固定的首选方法。如在详细描述别处16,18灌注必须在低压力下进行（例如，10厘米H 2 O），以避免压力相关灌注艺术品和损坏LSEC，典型表现内于细胞膜一样大的差距。然而，合理的固定通常可以使用从人类和动物的肝活检的针灌注获得，如在别处19进行详细描述。该技术包括直接注射入固定液的组织，直到血冲出样品和组织是坚定的和固定的。样品电子显微镜的固定需要被法律约束ormed尽快以下血流停止，以防止发生由于肝脏极其迅速自溶过程的结果超微结构改变。 我们还提出，最大限度地减少包含伪影，和标准化窗孔的测量图像分析的方法。变化血窦为显微照片的选择，人工制品的图像分析，并且小区区域的测量孔隙度和开窗频率已导致在已发表的结果重大差异。评估和窗孔和数据呈现的最低要求的测量标准化的做法没有得到明确解决在文献中以前4,10,20-31。

<table><tbody><tr><td>25% EM grade Glutaraldehyde</td><td>ProSciTech</td><td>C001</td><td>Store stock at -20 C until needed, avoid refreeze</td></tr><tr><td>Paraformaldehyde powder</td><td>Sigma Aldrich</td><td>158127</td><td>Always prepare Paraformaldehyde fresh</td></tr><tr><td>Sodium Cacodylate powder</td><td>Sigma Aldrich</td><td>C0250</td><td>Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH&lt;sub&gt;2&lt;/sub&gt;O, used mostly at 0.1 M by preparing 1:2 dilution</td></tr><tr><td>Calcium Chloride</td><td>Sigma Aldrich</td><td>C1016</td><td>Prepare 1 M CaCl2by dissolving powder in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Osmium tretroxide</td><td>ProSciTech</td><td>C011</td><td>Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td></tr><tr><td>Ethanol- Absolute</td><td>Sigma Aldrich</td><td>459836&amp;nbsp;</td><td>100 % Ethanol must be high grade and stored with Molecular Sieve</td></tr><tr><td>Other grades of Ethanol</td><td>Labtech</td><td>EL5</td><td>Prepare graded Ethanols with dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Hexamethyldisilazane</td><td>Sigma Aldrich</td><td>52619&amp;nbsp;</td><td>Allow to reach room temperature before use</td></tr><tr><td>Cannulas</td><td>Terumo</td><td>TSROX1832C, TSROX2225C, TSROX2419C</td><td>18 G is suitable for most rats, 22 G is suitable for most mice, but it is good to have a few 24 G on hand in case of very small mice</td></tr><tr><td>Conductive Carbon tape</td><td>ProSciTech</td><td>IA0201</td><td></td></tr><tr><td>Carbon Paint</td><td>ProSciTech</td><td>I003</td><td></td></tr><tr><td>Ketamine</td><td></td><td></td><td>Must be obtained under license</td></tr><tr><td>Xylazine</td><td></td><td></td><td>Must be obtained under license</td></tr><tr><td>Molecular Sieve</td><td>Sigma Aldrich</td><td>208647</td><td>Removes water from the 100 % Ethanol</td></tr></tbody></table>

a standardized method for the analysis of liver sinusoidal endothelial cells and their fenestrations by scanning electron microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

在低放大倍数的扫描电子显微镜初始可视揭示肝脏标本具有暴露面积大到足以观察许多大的肝血管和正弦波（ 图1A）的平坦表面。确保正确的肝块放置于安装存根是用于获得清晰的图像的正弦和 ​​肝脏的Glisson囊应避免这个原因（ 图1B）是必不可少的。增加放大率允许稠密脉管肝脏的仔细检查揭示该分割容器（ 图1C）的肝细胞的单细胞板。的肝导致图像质?...

Watch this Scientific Journal Video about A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy at JoVE.com

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

a-standardized-method-for-analysis-liver-sinusoidal-endothelial-cells

Research

JoVE Journal

Biology

14.4K 次观看.  University of Sydney and Concord Hospital. The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

视频: A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

科研

生物化学

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

癌症研究

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

免疫与感染

An Endothelial Planar Cell Model for Imaging Immunological Synapse Dynamics

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological synapses&#39;. Within these intimate cell-cell interfaces discrete sub-cellular clusters of MHC/Ag-TCR, F-actin, adhesion and signaling molecules form and remodel rapidly. These dynamics are thought to be critical determinants of both the efficiency and quality of the immune responses that develop and therefore of protective versus pathologic immunity. Current understanding of immunological synapses with physiologic APCs is limited by the inadequacy of the obtainable imaging resolution. Though artificial substrate models (e.g., planar lipid bilayers) offer excellent resolution and have been extremely valuable tools, they are inherently non-physiologic and oversimplified. Vascular and lymphatic endothelial cells have emerged as an important peripheral tissue (or stromal) compartment of &#39;semi-professional APCs&#39;. These APCs (which express most of the molecular machinery of professional APCs) have the unique feature of forming virtually planar cell surface and are readily transfectable (e.g., with fluorescent protein reporters). Herein a basic approach to implement endothelial cells as a novel and physiologic &#39;planar cellular APC model&#39; for improved imaging and interrogation of fundamental antigenic signaling processes will be described.

Adaptive immunity is controlled by dynamic 'immunological synapses' formed between T cells and antigen presenting cells. This protocol describes methods for investigating endothelial cells both as understudied physiologic APCs and as a novel type of 'planar cellular APC model'.

成像免疫突触动力学内皮平面细胞模型

Adaptive immunity is regulated by dynamic interactions between T cells and antigen presenting cells (&#39;APCs&#39;) referred to as &#39;immunological ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

生物学

Digestion of the Murine Liver for a Flow Cytometric Analysis of Lymphatic Endothelial Cells

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the lymph nodes where cellular debris and antigens can be surveyed. We are very interested in understanding how the lymphatic vasculature might be involved in inflammation and immune cell function within the liver. However, very little has been published establishing digestion protocols for the isolation of lymphatic endothelial cells (LECs) from the liver or specific markers that can be used to evaluate liver LECs on a per cell basis. Therefore, we optimized a method for the digestion and staining of the liver in order to evaluate the LEC population in the liver. We are confident that the method outlined here will be useful for the identification and isolation of LECs from the liver and will strengthen our understanding of how LECs respond to the liver microenvironment.

The goal of this protocol is to identify lymphatic endothelial cell populations within the liver using described markers. We utilize collagenase IV and DNase and a gentle mincing of tissue, combined with flow cytometry, to identify a distinct population of lymphatic endothelial cells.

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

Within the liver, lymphatic vessels are found within the portal triad, and their described function is to remove interstitial fluid from the liver to the ...

Visualizing Membrane Ruffle Formation using Scanning Electron Microscopy

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.

Macropinocytosis is a highly conserved endocytic process initiated by the formation of F-actin-rich sheet-like membrane projections, also known as membrane ruffles. Increased rate of macropinocytotic solute internalization has been implicated in various pathological conditions. This protocol presents a method to quantify membrane ruffle formation in vitro using scanning electron microscopy.

使用扫描电子显微镜可视化膜褶皱形成

Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may ...

医学

Using En Face Immunofluorescence Staining to Observe Vascular Endothelial Cells Directly

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of the intact endothelium will provide valuable information for understanding the cellular and molecular events in the dysfunctional endothelial cells. Here, we describe a modified en face immunofluorescence staining technique which enables scientists to obtain clear images of the intact endothelial surface and analyze the molecule expression patterns in situ. The method is simple and reliable for observing the entire endothelial monolayer at different sites of the aorta. This technique may be a promising tool for understanding the pathophysiology of atherosclerosis, especially at an early stage.

Here, we present a protocol for immunofluorescence staining to observe the endothelial cells of the mouse aorta directly. This technique is useful when studying the cellular and molecular phenotype of endothelial cells in different flow patterns and in the development of atherosclerosis.

使用 En 面免疫荧光染色直接观察血管内皮细胞

Aberrant changes in endothelial phenotype and morphology are considered to be initial events in the pathogenesis of atherosclerosis. Direct observation of ...

Serial Block-Face Scanning Electron Microscopy (SBF-SEM) of Biological Tissue Samples

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, offering an unprecedented three-dimensional view of tissue microanatomy. While SBF-SEM has seen an exponential increase in use in recent years, technical aspects such as proper tissue preparation and imaging parameters are paramount for the success of this imaging modality. This imaging system benefits from the automated nature of the device, allowing one to leave the microscope unattended during the imaging process, with the automated collection of hundreds of images possible in a single day. However, without appropriate tissue preparation cellular ultrastructure can be altered in such a way that incorrect or misleading conclusions might be drawn. Additionally, images are generated by scanning the block-face of a resin-embedded biological sample and this often presents challenges and considerations that must be addressed. The accumulation of electrons within the block during imaging, known as "tissue charging," can lead to a loss of contrast and an inability to appreciate cellular structure. Moreover, while increasing electron beam intensity/voltage or decreasing beam-scanning speed can increase image resolution, this can also have the unfortunate side effect of damaging the resin block and distorting subsequent images in the imaging series. Here we present a routine protocol for the preparation of biological tissue samples that preserves cellular ultrastructure and diminishes tissue charging. We also provide imaging considerations for the rapid acquisition of high-quality serial-images with minimal damage to the tissue block.

Serial Block-Face Scanning Electron Microscopy SBF-SEM of Biological Tissue Samples

This protocol outlines a routine method for using serial block-face scanning electron microscopy (SBF-SEM), a powerful 3D imaging technique. Successful application of SBF-SEM hinges on proper fixation and tissue staining techniques, as well as careful consideration of imaging settings. This protocol contains practical considerations for the entirety of this process.

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

Serial block-face scanning electron microscopy (SBF-SEM) allows for the collection of hundreds to thousands of serially-registered ultrastructural images, ...

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

小鼠原发性肝正弦内皮细胞的分离与表征

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

摘要

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此视频中的章节

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

成像免疫突触动力学内皮平面细胞模型

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

使用扫描电子显微镜可视化膜褶皱形成

使用 En 面免疫荧光染色直接观察血管内皮细胞

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）

小鼠原发性肝正弦内皮细胞的分离与表征

一个标准化的方法肝窦内皮细胞的分析及其窗孔用扫描电子显微镜

摘要

探索更多视频

此视频中的章节

小鼠肝灌注肝细胞和正弦内皮细胞的纯化

斑马鱼非酒精性脂肪肝相关肝细胞癌早期进展期间的肝脏微环境分析

一个流程粘附实验来研究白细胞招聘剪切应力的人力肝窦内皮细胞条件下

成像免疫突触动力学内皮平面细胞模型

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

小鼠肝脏消化对淋巴内皮细胞的流式细胞仪分析

使用扫描电子显微镜可视化膜褶皱形成

使用 En 面免疫荧光染色直接观察血管内皮细胞

生物组织样本的串行块面扫描电子显微镜 （SBF-SEM）

小鼠原发性肝正弦内皮细胞的分离与表征

生物组织样本的串行块面扫描电子显微镜（SBF-SEM）