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

12.4K 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-α in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

实时数字成像大鼠提睾肌缺血再灌注损伤（IRI）的白细胞 - 内皮细胞相互作用

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

科研

医学

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in tumorigenesis, can be directed to cancer biomarkers enabling tumor detection and characterization, and can be used for cancer therapy as mAbs or antibody-drug conjugates to activate immune effector cells, to inhibit signaling pathways, or directly kill cells carrying the specific antigen. Despite clinical advancements in the development and production of novel and highly specific mAbs, diagnostic and therapeutic applications can be impaired by the complexity and heterogeneity of the tumor microenvironment. Thus, for the development of efficient antibody-based therapies and diagnostics, it is crucial to assess the biodistribution and interaction of the antibody-based conjugate with the living tumor microenvironment. Here, we describe In Vivo Immunofluorescence Localization (IVIL) as a new approach to study interactions of antibody-based therapeutics and diagnostics in the in vivo physiological and pathological conditions. In this technique, a therapeutic or diagnostic antigen-specific antibody is intravenously injected in&#160;vivo and localized ex vivo with a secondary antibody in isolated tumors. IVIL, therefore, reflects the in vivo biodistribution of antibody-based drugs and targeting agents. Two IVIL applications are described assessing the biodistribution and accessibility of antibody-based contrast agents for molecular imaging of breast cancer. This protocol will allow future users to adapt the IVIL method for their own antibody-based research applications.

The in vivo immunofluorescence localization (IVIL) method can be used to examine in vivo biodistribution of antibodies and antibody conjugates for oncological purposes in living organisms using a combination of in vivo tumor targeting and ex vivo immunostaining methods.

在Vivo免疫荧光定位中用于评估癌症研究中的治疗和诊断抗体生物分布

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

癌症研究

Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn's Disease using Immunofluorescence

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis disorder and damage of intestinal mucosa and seems to be associated with an increased risk of colon neoplastic transformation. Accumulating evidence indicates that estrogens and estrogen receptors affect not only hormone-sensitive tissues, but also other tissues not directly related to estrogens, such as the lungs or colon. Here, we describe the protocol for the successful immunofluorescence staining of estrogen receptors in colon obtained from a murine model of TNBS-induced Crohn&#39;s disease. A detailed protocol for the induction of Crohn&#39;s disease in mice and intestine preparation is provided as well as a step-by-step immunohistochemical procedure using formalin-fixed paraffin-embedded intestine sections. The described methods are not only useful for estrogen receptor detection and estrogen signaling investigation in vivo but can also be applied to for other proteins which may be involved in the development of colitis.

The protocol presents a complete validated TNBS-induced murine model of Crohn&#39;s disease and methods for visualization of estrogen receptors by immunohistochemistry using immunofluorescence of formalin-fixed colon sections embedded in paraffin.

使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化

Crohn&#39;s disease is the most diagnosed type of inflammatory bowel disease. Chronic inflammation developing in the intestine leads to peristalsis ...

生物化学

Using 16&#945;-[18F]-Fluoro-17&#946;-Estradiol PET to Visualize Estrogen Receptor &#945; Expression in Human Breast Cancer Xenografts in Female Ovariectomized Mice

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET), ovariectomized BALB/c nude mice were injected with ER&#945;-positive breast cancer cells (MCF-7, 3 &#215; 106 cells; shoulder [n = 10] or 4th inguinal mammary fat pad [n = 10]) or ER&#945;-negative breast cancer cells (MDA-MB-231, 1 &#215; 106 cells; mammary fat pad [n = 5]). Mice harboring MCF-7 cells received subcutaneous injections of 20 &#181;g of 17&#946;-estradiol (20 &#181;g/20 &#181;L; corn oil:ethanol, 9:1) in the nape of their necks 2 days prior to cell injection, followed by daily injections five times per week for 5 weeks. Tumor volumes were measured according to the formula: (L*W2)/2 (L; length, W; width). Once tumor volumes reached approximately 100 mm3, 17&#946;-estradiol injections were halted 2 days prior to mice receiving 18F-FES for PET imaging to avoid competitive binding with ER&#945;. Upon 18F-FES administration via the lateral tail vein, PET/MRI was performed for 15 min at 1 h to 1.5 h post-injection. 18F-FES uptake was not observed in ER&#945;-negative, MDA-MB-231 tumor-bearing mice. 18F-FES uptake was most pronounced in mice harboring MCF-7 tumors in the shoulder. In MCF-7 tumors grown in the inguinal mammary fat pad, 18F-FES uptake was less visible, as the intestinal excretion pattern of 18F-FES obscured the radioactivity detectable in these tumors. To use 18F-FES PET as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts, we demonstrate that the visibility of 18F-FES uptake is clear in tumors located away from the abdominal region of mice, such as in the shoulder.

Here, we demonstrate a protocol to use 16&#945;-[18F]-fluoro-17&#946;-estradiol (18F-FES) positron emission tomography (PET) as a tool to visualize ER&#945; expression in ER&#945;-positive breast xenografts.

使用 16α-[18F]-氟-17β-雌二醇 PET 可视化雌性卵巢切除小鼠人乳腺癌异种移植物中雌激素受体α表达

To demonstrate how estrogen receptor alpha (ER&#945;) positive breast cancer xenografts may be visualized in BALB/c nude mice using ...

Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping.
Tissue specimens are harvested and stored at -80 &#176;C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in 125I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: &#39;non-specific binding&#39; (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and &#39;total binding&#39; with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 &#181;M) is also present in the incubation buffer to limit 125I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 &#176;C, NSP slides are exposed to 10 &#181;M PD123319 and losartan, while &#39;total binding&#39; slides are exposed to 10 &#181;M PD123319. Slides are then incubated with 125I-SI-Ang II in the presence of PD123319 for &#39;total binding&#39;, and PD123319 and losartan for NSP in assay buffer, followed by several &#39;washes&#39; in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons.
The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

Here we present a protocol to describe the localization of angiotensin II Type 1 receptors in the rat brain by quantitative, densitometric, in vitro receptor autoradiography using an iodine-125 labeled analog of angiotensin II.

受体放射自显影协议血管紧张素Ⅱ受体的本地化的可视化

This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to ...

神经科学

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

大鼠肠系膜外化:调查涉及在血管生成细胞动力学模型

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

生物学

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

细胞内钙的双光子成像<sup&gt; 2+</sup&gt;的离体大鼠主动脉的处理和一氧化氮的内皮细胞和平滑肌细胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

视网膜低温切片、全支架和低渗隔离血管毛细血管微血管组织免疫组化的制备

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

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 ...

Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.

This article provides a detailed methodology for the measurement of isolevuglandins in tissues by immunofluorescence using alkaline phosphatase-conjugated ScFv D11 antibody. Hypertension models in both mice and humans are used to explain the step-by-step procedures and fundamental principles associated with isolevuglandin measurement in tissue samples.

使用 D11 scFv-碱性磷酸酶融合蛋白和免疫荧光直接检测组织中的异左旋蛋白

Isolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to ...