The authors have no acknowledgements.

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>

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Name of material</td>
			<td style="width:100px;">Company</td>
			<td style="width:93px;">Catalogue Number</td>
			<td style="width:311px;">Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">EM grade Glutaraldehyde</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C001</td>
			<td style="width:311px;">Store stock at -20 C until needed, avoid refreeze</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Paraformaldehyde powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>158127</td>
			<td style="width:311px;">Always prepare Paraformaldehyde fresh</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:155px;">Sodium Cacodylate powder</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C0250</td>
			<td style="width:311px;">Prepare 0.2 M stock, pH 7.4 by dissolving powder in dH2O, used mostly at 0.1 M by preparing 1:2 dilution</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:155px;">Calcium Chloride</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">C1016</td>
			<td style="width:311px;">Prepare 1 M CaCl2by dissolving powder in dH2O</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:155px;">Osmium tretroxide</td>
			<td style="width:100px;">ProSciTech</td>
			<td style="width:93px;">C011</td>
			<td style="width:311px;">Wash ampoules in weak acid prior to use to avoid contamination. Prepare 2 % stock in glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:155px;">Ethanol- Absolute</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>459836&#160;</td>
			<td style="width:311px;">100 % Ethanol must be high grade and stored with Molecular Sieve</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Other grades of Ethanol</td>
			<td style="width:100px;">Labtech</td>
			<td style="width:93px;">EL5</td>
			<td style="width:311px;">Prepare graded Ethanols with dH2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Hexamethyldisilazane</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td>52619&#160;</td>
			<td style="width:311px;">Allow to reach room temperature before use</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:155px;">Cannulas</td>
			<td style="width:100px;">Terumo</td>
			<td style="width:93px;">TSROX1832C, TSROX2225C, TSROX2419C</td>
			<td style="width:311px;">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 height="42">
			<td height="42" style="height:42px;width:155px;">Conductive Carbon tape</td>
			<td style="width:100px;">ProSciTech</td>
			<td>IA0201</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Carbon Paint</td>
			<td style="width:100px;">ProSciTech</td>
			<td>I003</td>
			<td style="width:311px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Ketamine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be optained under licence</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Xylazine</td>
			<td style="width:100px;"></td>
			<td style="width:93px;"></td>
			<td style="width:311px;">Must be obtained under licence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:155px;">Molecular Sieve</td>
			<td style="width:100px;">Sigma Aldrich</td>
			<td style="width:93px;">208647</td>
			<td style="width:311px;">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

14.2K 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.