这个项目的工作是由来自美国国立卫生R01 - NR5339研究院的资助和统一服务大学健康科学R061LD的支持。

1。软脑膜动脉的分离和制备<ol><li>虽然大鼠麻醉，插入导管进入动脉灌注1％伊文思蓝在1X PBS安全。软脑膜血管染色以及与伊文思蓝染料使它们更容易被孤立。 </li><li>提取在-70℃直至所需的大脑和存储。理想情况下，大脑应存放不超过2个月。 </li><li>使用解剖显微镜去除大脑表面的脑膜动脉（平均直径35-50微米）。从背侧和外侧的皮质表面使用细嘴镊子小心拉出所有的蓝染动脉。之前，取出放置在固定液壁多余的皮层组织。 </li><li>修正了在2％冷多聚甲醛在0.1M PBS 30分钟的收获动脉。 </li><li>为了准备切片的动脉倒在冻结盘/夹头（-20℃）包埋剂。 </li><li>广场动脉部分夹头一个平面ND等待它冻结。牢固的地方船只，右的低温恒温器冻结盘上，你面临的动脉尖嵌入媒体。用镊子握住它，直到它扎实冻结。 </li><li>与血管冻结光盘放置到低温恒温器头，并切断与低温恒温器8微米软脑膜动脉环。 </li><li>山铬钾明胶subbed幻灯片的动脉。 </li><li>存放在冰箱准备的幻灯片在幻灯片中，在4 ° C过夜。 </li></ol> 2。 ER -α的免疫荧光染色第1天<ol><li>从冰箱中取出的幻灯片;带给每到室温。 </li><li>洗幻灯片倒入1-1.5毫升0.1M PBS（足以覆盖所有的部分），让我们坐了10分钟，漏和重复两次以上的过程。每个幻灯片的边缘有一个疏水性的标记。因此，液体仍然是表面上的幻灯片。随着各洗涤液浇出来，用新鲜的0.1 M PBS代替。 </li><li>孵育1毫升在室温下30分钟的50毫米氯化铵的幻灯片，以减少内源性荧光。 </li><li>在0.1M PBS洗3x10分钟的幻灯片。描述在2.2 </li><li>座0.1％TRITON - X 100加1％正常山羊血清（NGS）的PBS 30分钟的幻灯片。以减少非特异性结合。 </li><li>孵育一抗（兔抗ER -α; 1:500）样品在PBS + 0.1％的Triton - X + 1％农工商一夜之间在4 ° C。 </li></ol>第2天<ol start="7"><li>从冰箱中取出的幻灯片;带给每到室温。 </li><li>在0.1M PBS洗3x10分钟的幻灯片。在2.2中描述</li><li>俄勒冈绿488中学抗兔农工商1％，0.1％TRITON - X100 + 2小时，PBS在室温避光孵育。这一步，​​接下来的步骤必须在黑暗中。 </li><li>在0.1M PBS洗3x10分钟的幻灯片。在2.2中描述</li><li>根据一个VEntilated罩，放置1 4&#39;下降，6 diamidino - 2 -苯基吲哚（DAPI）加安装媒体上的船只，然后放置在样品的盖玻片。 </li><li>应用透明指甲油密封盖玻片边缘。不要移动，直到完全干燥的幻灯片，这大约需要24小时。 </li></ol> 3。数字荧光成像<ol><li>成像在软脑膜血管ER -α片，我们使用尼康的Eclipse 80I数字荧光显微镜配备一台数码相机的3种颜色（蓝色，绿色和红色）的过滤器。 </li><li>与染色在显微镜下的软脑膜血管段的插入准备的幻灯片和调整，以可视化的利益荧光标记的地区。 X100放大倍率开始，然后增加X600的放大倍率。 </li><li>使用的相机，捕捉到的DAPI（蓝色）FITC（绿色）细胞核（DAPI）和ER -α（俄勒冈州格林），运用了尼康扩展深入的重点软件渠道图像转换多个视图细分船只进入2 - D图像。 </li></ol> 4。代表性的成果： 我们本地化的雌激素受体-α在使用荧光探针的软脑膜动脉血管和优化使用数字荧光显微镜受体的可视化。要检测的受体，多克隆抗体和俄勒冈绿488标记的第二抗体用于形象的受体在大脑表面隔离的脑膜动脉存在。另外，核染色（DAPI）的用于识别细胞核，并确定如果我们能够探测位于细胞核中的受体，因为ER -α的细胞cyotosol和细胞核中有报道。此外，我们进行了验证性实验来验证的特异性抗体，来验证的二次抗体结合。 <img src="/files/ftp_upload/3203/3203fig1.jpg" alt="图1"/><br/&gt; 图1免疫荧光染色为ER -α（绿色）和雌性大鼠脑隔离在软脑膜动脉环用DAPI（蓝色）counterstaining。图1A显示合并后的图像表明存在的一些核内雌激素受体（箭头）。图1B和1C集中的ER -α的形象，这是一个组合Z - Stack的图像。 X600放大倍率和图像，是从同一船只在不同的平面切割环。箭头表明雌激素受体α。 <img src="/files/ftp_upload/3203/3203fig2.jpg" alt="图2"/> 图2对照组雌性大鼠脑隔离在软脑膜动脉环的免疫荧光染色。图2D显示没有主要抗体的船只。图2E显示无二次抗体（注意不同的自体荧光沿内皮方）的船只。图像X600放大倍率和切环在同一船只differeNT飞机。

<ol>
	<li>Smiley, D. A., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+compounds,+estrogen+receptors+and+vascular+cell+signaling+in+the+aging+blood+vessels.">Estrogen compounds, estrogen receptors and vascular cell signaling in the aging blood vessels.</a> Curr. Med. Chem. 16, 1863-1887 (2009).</li><li>Miller, V. M., Duckles, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+actions+of+estrogens:+functional+implications.">Vascular actions of estrogens: functional implications.</a> Pharmacol. Rev. 60, 210-241 (2008).</li><li>Duckles, S. P., Krause, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+effects+of+oestrogen:+multiplicity+of+action.">Cerebrovascular effects of oestrogen: multiplicity of action.</a> Clin. Exp. Parmacol. Physiol. 34, 801-808 (2007).</li><li>Stirone, C., Duckles, S. P., Krause, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+forms+of+estrogen+receptor-a+incerebral+blood+vessels:+regulation+by+estrogen.">Multiple forms of estrogen receptor-a incerebral blood vessels: regulation by estrogen.</a> Am. J. Physiol. Endocrinol. Metab. 284, E184-E192 (2003).</li><li>Dan, P., Cheung, J. C., Scriven, D. R., Moore, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope-dependent+localization+of+estrogen+receptor-alpha,+but+not+-beta,+in+en+face+arterial+endothelium.">Epitope-dependent localization of estrogen receptor-alpha, but not -beta, in en face arterial endothelium.</a> Am. J. Physiol. Heart Circ. Physiol. 284, 1295-1306 (2003).</li><li>Fritschy, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+my+antibody+staining+specific?+How+to+deal+with+pitfalls+of+immunohistochemistry.">Is my antibody staining specific? How to deal with pitfalls of immunohistochemistry.</a> Eur. J. Neurosci. 28, 2365-2370 (2008).</li><li>Watanabe, Y., Littleton-Kearney, M. T., Traystman, R. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+restores+post-ischemic+pial+and+microvascular+dilation.">Estrogen restores post-ischemic pial and microvascular dilation.</a> Am. J. Physiol. Heart and Circ. 281, H155-H160 (2001).</li><li>Li, M., Zeynalov, E., Li, X., Miyazaki, C., Koehler, R. C., Littleton-Kearney, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+estrogen+on+postischemic+pial+artery+reactivity+to+ADP.">Effects of estrogen on postischemic pial artery reactivity to ADP.</a> Microcirculation. 16, 403-413 (2009).</li><li>Qin, X. Y., Hurn, P. D., Littleton-Kearney, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+restores+postischemic+sensitivity+to+thromboxane+mimetic+U46619+in+rat+pial+artery.">Estrogen restores postischemic sensitivity to thromboxane mimetic U46619 in rat pial artery.</a> Cereb. Blood. Flow. Metab. 25, 1041-1046 (2005).</li><li>Miyazaki, C., Koelher, R. C., Littleton-Kearney, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+HET0016+and+estrogen+on+pial+artery+vasodilatory+capacity.">Effects of HET0016 and estrogen on pial artery vasodilatory capacity.</a> FASEBJ Abstracts. , (2007).</li><li>Daneshtalab, N., Dore, J. J. E., Smeda, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: procedures in immunohistochemical studies.</a> J. Pharmacol. Toxical. Methods. 61, 127-135 (2010).</li><li>Lorincz, A., Nusser, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specificity+of+immunoreactants.">Specificity of immunoreactants.</a> J. Neurosci. 28, 9083-9086 (2008).</li></ol>

所表达的意见是作者的，并不能反映官方政策或卫生科学统一服务大学，国防部，或美国政府的立场。

在此之前，我们发现，短暂性缺血性脑损伤后软脑膜动脉的能力，改变直径都扩血管7，第 8和血管收缩剂9明显是在年轻的去势大鼠抑郁。慢性雌激素饱食恢复血管舒缩反应7-9。此外，雌激素替代延迟反转软脑膜动脉血管舒张能力领导我们的问题是否长期的雌激素的枯竭影响的cerebrovasculature ER -α 密度 10雌激素的有益作用。我们计划用免疫荧光标记结合免疫印迹评估慢性雌激素耗尽ER -α表达的变化。因为我们有一些可视化的受体离散群体的困难，我们修改的方法，我们在这里展示。因此，我们在这项研究的主要目标是首先制定一个相对简单的的方法来优化视觉化的软脑膜动脉的受体。在几份报告协议6,11,12中，我们发现有必要作调整的组织类型，我们探测，样品的厚度，ER -α在血管分布，以及非特异性结合的程度。 我们的方法是非常有效的可视化的ER -α在软脑膜血管切片的存在。但其他人曾指出，这些方法的成功运用是高度依赖的特异性和浓度的抗体，利息，固定和阻塞协议和组织处理 12的蛋白质的亚细胞定位。我们的船准备的可视化之前，我们所花费的时间工作，所有这些变量。我们强调如何在本文中，我们需要调整我们的一些做法。 我们的目标是最佳的可视化在软脑膜动脉ER -α，并提出了一些挑战。首先，大鼠软脑膜动脉的平均规模为35-50微米隔离和消除脑表面有些棘手之间。我们发现，牺牲前动脉夹层动脉瘤容易与伊文思蓝染料灌注。可以影响埃文蓝色荧光，但我们认为，使用这种染料的优势超过了它的小缺点。在我们的协议的船只被冲几次。这可以最大限度地减少在船只的残余染料和荧光染料的减少可能造成的影响。我们遇到的另一个困难是软脑膜血管段的厚度作出清晰的可视化的受体困难。此外，我们还无法获得清晰的图像，尤其是可能是由于整个几个细胞层分散，ER -α。我们试图与纵向血管切片（如其他人）的工作，但由于软脑膜大小小，难以处理的船只，防止扭曲的船只，而安装在幻灯片上使用显微镜。最后，我们成功地修改了我们的技术，通过使用一个恒温器和削减至8微米厚环的船只跨明智。环好办了几个可以安装在一个幻灯片。 一旦我们收购的船只，我们认为有必要测试第一闪光灯，然后固定船只Danseshatalb及联营公司11建议冻结组织的有效性。虽然我们发现轻微不足的背景免疫，我们也注意到，受体染色不太激烈的可视化的受体更加困难。因此，我们宁可先冻结整个大脑和存储（一般在1-2个月），直到需要。然后，我们拉断所述的船只和修复血管切片的低温恒温器前。 ，因为我们在这里展示。 一个必要的步骤是小学和中学抗体分别与运行控制，确定特异性和背景免疫。雌激素的受体α抗体可困难的工作。事实上，我们尝试了3种不同的的抗体（1单克隆抗体和多克隆2个）之前，我们与我们的受体染色的成功感到满意。作为我们控制的一部分，我们首先验证抗体的特异性抗体此外省略。图2D显示船只无一抗。有没有染色和一点点的背景信号。由于是多克隆抗体，有一些弥漫性非特异性背景染色。其次，我们的主要抗体，但没有二次抗体（图2E）。人们可以看到一个不错的利润率，沿血管内皮边界autofluoresence。丹和联营公司5的工作，这是一致的。然而，既不免疫荧光的小点代表的ER -α，也不是颗粒状的外观文献lecting众多受体的存在，可以检测没有小学和中学的抗体相结合。我们注意到，沿血管内皮利润率autofluoresence不同的模式。抗体的特异性，这些独立的测试是很重要的，因为他们确认为ER -α特异性抗体和二级抗体绑定到主。 最后，在其他4表明，5脑血管含有大量雌激素受体​​α的分型受体。在我们手中，准备从8微米的横截面切片孤立的软脑膜动脉，提高染色和可视化的受体。传统的方法使用共聚焦显微镜研究样品。有能力从串行光切片厚标本共聚焦显微镜是一个重要的好处。然而，购买一个良好的共聚焦显微镜可以是非常昂贵。通过使用扩展深度（EDF）软件，我们发现，可以减少我们的设备成本。我们使用荧光显微镜与EDF获得清晰的图像，并能够可视化使用Z -堆栈的几个层次上的软脑膜动脉的ER -α。法国电力软件的一个显着的好处是，它允许的方式，有效地创建一个从图像中的3 - D图像采集。对于没有访问共聚焦显微镜或资金购买一台数字拨软件荧光显微镜的实验室可能是一个实用的，成本更低的选择，取决于研究者的目标。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>伊文思蓝</td><td>西格玛</td><td> E - 2129 </td></tr><tr><td> PBS 10X </td><td>西格玛</td><td> P - 5493 </td></tr><tr><td>多聚甲醛</td><td> Fisher Scientific则</td><td> T353 </td></tr><tr><td>组织TEK华侨城复合</td><td> VWR </td><td> 25608-930 </td></tr><tr><td>氯化铵</td><td>西格玛</td><td> A9434 </td></tr><tr><td> TRITON X - 100 </td><td>西格玛</td><td> T9284 </td></tr><tr><td>正常山羊血清</td><td> Invitrogen公司</td><td> 16210-064 </td></tr><tr><td> ER -α多克隆抗体</td><td>圣克鲁斯</td><td> H - 184 </td></tr><tr><td>俄勒冈州绿色488羊抗兔IgG </td><td> Invitrogen公司</td><td> O - 11038 </td></tr><tr><td> Vectashield安装培养基 荧光用DAPI </td><td>媒介实验室</td><td> H - 1200 </td></tr></tbody></table>

imaging of estrogen receptor-&alpha; in rat pial arterioles using a digital immunofluorescent microscope

许多雌激素对血管反应性的影响是通过相互作用介导雌激素受体​​1，2，3 。虽然存在两个子类型（雌激素受体​​-α和β），雌激素受体​​α已确定在平滑肌和内皮细胞的软脑膜动脉段，采用荧光染色结合激光扫描共聚焦显微镜 4 。此外，ER -α是位于细胞核和细胞质大鼠基底动脉5。丰富的受体和荧光明亮，但受体离散群体清晰的可视化是很难的，可能由于许多细胞层软脑膜血管段位于的数目。此外，采用免疫组化技术的许多报道搭配共聚焦显微镜不佳详细的要求再现实验 6的关键。我们对这篇文章的目的是描述一个简单的方法来优化吨HE染色和可视化的ER -α使用软脑膜动脉横截面切片获得雌性大鼠脑。我们首先与伊文思蓝染料灌注大鼠可以轻松地找到我们在解剖显微镜下分离表面的软脑膜动脉。使用一个恒温器片8微米的血管截面，让我们获得薄的船只部分，所以，不同的船只飞机都更清楚地可视化。整个船只，而不是使用一个小的血管段切割，更容易观看的内皮细胞和平滑肌层的优势。此外，用免疫荧光显微镜数字扩展深入软件生产十到十二种不同的船只飞机的清晰图像，并且成本低于共聚焦激光扫描显微镜的使用。

Watch this Scientific Journal Video about Imaging of Estrogen Receptor-Î± in Rat Pial Arterioles using a Digital Immunofluorescent Microscope at JoVE.com

Imaging of Estrogen Receptor-&amp;#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

这篇文章的目的是证明的方法，优化免疫检测雌激素受体-α（ERα）​​使用数字免疫荧光显微镜在大鼠的软脑膜动脉切片。

使用数字免疫荧光显微镜成像在大鼠软脑膜小动脉雌激素受体α

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

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Biology

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

12.3K 次观看.  Uniformed Services University of the Health Sciences. 这篇文章的目的是证明的方法，优化免疫检测雌激素受体-α（ERα）​​使用数字免疫荧光显微镜在大鼠的软脑膜动脉切片。

视频: Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Proper Care and Cleaning of the Microscope

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a microscope causes reduction in contrast and resolution. DIC is especially sensitive to contamination and scratches on the lens surfaces. This protocol details the procedure for keeping the microscope clean.

适当的照顾和清洁的显微镜

Keeping the microscope optics clean is important for high-quality imaging. Dust, fingerprints, excess immersion oil, or mounting medium on or in a ...

科研

生物学

Major Components of the Light Microscope

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for different applications, and how it should be maintained as required to obtain maximum image-forming capacity and resolution. The components of the microscope are described in detail here.

光镜下的主要组成部分

The light microscope is a basic tool for the cell biologist, who should have a thorough understanding of how it works, how it should be aligned for ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

成像 C。线虫胚胎使用啶显微镜和开放源码软件

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces photo-bleaching and phototoxic effects compared to confocal microscopy, while providing 3D images with subcellular resolution. Here we present the setup of a light sheet based microscope, which is composed of an upright microscope and a small set of opto-mechanical elements for the generation of the light sheet. The protocol describes how to build, align the microscope and characterize the light sheet. In addition, it details how to implement the method for in toto imaging of C. elegans embryos using a simple observation chamber. The method allows the capture of 3D two-colors time-lapse movies over few hours of development. This should ease the tracking of cell shape, cell divisions and tagged proteins over long periods of time.

Setting Up a Simple Light Sheet Microscope for In Toto Imaging of C. elegans Development

This protocol describes the setup of a light sheet microscope and its implementation for in vivo imaging of C. elegans embryos.

设置简单的轻表为显微镜<em&gt;在托托</em的&gt;成像<em&gt; C。线虫</em&gt;发展

Fast and low phototoxic imaging techniques are pre-requisite to study the development of organisms in toto. Light sheet based microscopy reduces ...

Implementation of a Coherent Anti-Stokes Raman Scattering (CARS) System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become available for biologists. These systems are primarily designed for multi-channel two-photon fluorescence microscopy. However, without any modification, complementary non-linear optical microscopy such as second-harmonic generation (SHG) or third harmonic generation (THG) can also be performed with this set-up, allowing label-free imaging of structured molecules or aqueous medium-lipid interfaces. These techniques are well suited for in-vivo observation, but are limited in chemical specificity. Chemically selective imaging can be obtained from inherent vibration signals based on Raman scattering. Confocal Raman microscopy provides 3D spatial resolution, but it requires high average power and long acquisition time. To overcome these difficulties, recent advances in laser technology have permitted the development of nonlinear optical vibrational microscopy, in particular coherent anti-Stokes Raman scattering (CARS). CARS microscopy has therefore emerged as a powerful tool for biological and live cell imaging, by chemically mapping lipids (via C-H stretch vibration), water (via O-H stretch vibrations), proteins or DNA. In this work, we describe the implementation of the CARS technique on a standard OPO-coupled multiphoton laser scanning microscope. It is based on the in-time synchronization of the two laser lines by adjusting the length of one of the laser beam path. We present a step-by-step implementation of this technique on an existing multiphoton system. A basic background in experimental optics is helpful and the presented system does not require expensive supplementary equipment. We also illustrate CARS imaging obtained on myelin sheaths of sciatic nerve of rodent, and we show that this imaging can be performed simultaneously with other nonlinear optical imaging, such as standard two-photon fluorescence technique and second-harmonic generation.

Implementation of a Coherent Anti-Stokes Raman Scattering CARS System on a Ti:Sapphire and OPO Laser Based Standard Laser Scanning Microscope

Coherent anti-Stokes Raman scattering (CARS) microscopy based on inherent vibration of molecule bonds permits label-free chemically selective live cell imaging. This work presents the implementation of a complementary microscopy technique on a standard multiphoton laser scanning microscope based on a femtosecond Ti:sapphire laser and an OPO laser.

蓝宝石OPO基于激光的标准激光扫描显微镜：在TI相干反斯托克斯拉曼散射（CARS）系统的实现

Laser scanning microscopes combining a femtosecond Ti:sapphire laser and an optical parametric oscillator (OPO) to duplicate the laser line have become ...

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

在大鼠提睾肌的小动脉的可视化和量化的无细胞层

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine disruption. Estrogenic ligands include both natural and manmade &#34;Environmental Estrogens&#34; (EEs) found in many consumer goods including Personal Care Products (PCPs), plastics, pesticides, and foods. EEs disrupt hormone signaling in humans and other animals, potentially reducing fertility and increasing disease risk. Despite the importance of EEs and other Endocrine Disrupting Chemicals (EDCs) to public health, endocrine disruption is not typically included in undergraduate curricula. This shortcoming is partly due to a lack of relevant laboratory activities that illustrate the principles involved while also being accessible to undergraduate students. This article presents an optimized YES for quantifying ligands in personal care products that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates one of the two colorimetric substrates (ortho-nitrophenyl-&#946;-D-galactopyranoside (ONPG) or chlorophenol red-&#946;-D-galactopyranoside (CPRG)) that are cleaved by &#946;-galactosidase, a 6-day refrigerated incubation step to facilitate use in undergraduate laboratory courses, an automated application for LacZ calculations, and R code for the associated 4-parameter logistic regression analysis. The protocol has been designed to allow undergraduate students to develop and conduct experiments in which they screen products of their choosing for estrogen mimics. In the process, they learn about endocrine disruption, cell culture, receptor binding, enzyme activity, genetic engineering, statistics, and experimental design. Simultaneously, they also practice fundamental and broadly applicable laboratory skills, such as: calculating concentrations; making solutions; demonstrating sterile technique; serially diluting standards; constructing and interpolating standard curves; identifying variables and controls; collecting, organizing, and analyzing data; constructing and interpreting graphs; and using common laboratory equipment such as micropipettors and spectrophotometers. Thus, implementing this assay encourages students to engage in inquiry-based learning while exploring emerging issues in environmental science and health.

This article presents an optimized yeast estrogen screen for quantifying ligands in Personal Care Products (PCPs) that bind estrogen receptors alpha (ER&#945;) and/or beta (ER&#946;). The method incorporates two colorimetric substrate options, a six-day refrigerated incubation for use in undergraduate courses, and statistical tools for data analysis.

应用酵母雌激素筛检优选本科教学实验室的个人护理产品中的雌激素配体

The Yeast Estrogen Screen (YES) is used to detect estrogenic ligands in environmental samples and has been broadly applied in studies of endocrine ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

基于 X 射线 Microcomputed 层析成像的玉米血管束微米尺度分型技术研究

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure for primary hepatocyte isolation is technically challenging, especially in portal vein cannulation. The procedure is also prone to occasional contamination and variations in perfusion conditions due to difficulties in the assembly, optimization, or maintenance of the perfusion setup. Here, a detailed protocol for an improved two-step collagenase perfusion procedure with multiparameter perfusion control is presented. Primary rat hepatocytes were successfully and reliably isolated by taking the necessary technical precautions at critical steps of the procedure, and by reducing the operational difficulty and mitigating the variability of perfusion parameters through the adoption of a special intravenous catheter, standardized sterile disposable tubing, temperature control, and real-time monitoring and alarm system. The isolated primary rat hepatocytes consistently exhibit high cell viability (85%-95%), yield (2-5 x 108 cells per 200-300 g rat) and functionality (albumin, urea and CYP activity). The procedure was complemented by an integrated perfusion system, which is compact enough to be set up in the laminar flow hood to ensure aseptic operation.

This protocol details the use of a special intravenous catheter, standardized sterile disposable tubing, temperature control complemented by real-time monitoring, and an alarm system for two-step collagenase perfusion procedure to improve the consistency in the viability, yield, and functionality of isolated primary rat hepatocytes.

多参数灌注对照分离原代大鼠肝细胞

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...