这项工作是由美国心脏协会（EIA 0740129N）和NIH的T32 HL90610支持。

1。实验前<ol><li>实验一天前，站适当尺寸的玻璃毛细管拉成微电极（无论是水平或垂直的车夫，都可以使用）。尖端直径可以根据被隔离的船只随时调整，虽然我们通常使用直径范围50-150微米之间。这些微血管站了以下丁烷火焰加热到适当的配置，然后弯曲。微量提示身体打破近似直径（用细镊子）微血管中的问题，可以适当的构象，如针对特定应用所需的研磨或抛光。读者对这些程序的广泛和出色的审查，导演戴维斯等。16。两个微电极，然后放置在反对微血管室移液器持有人。这些必须面向这样的提示在相同的水平和垂直平面内的血管腔。 在这个手稿（ 图1和2）微血管腔是一个建先生，David Eick威斯康星医学院生理学教研室（定制<a href="http://www.phys.mcw.edu/">http://www.phys.mcw.edu/</a>和<a href="http://www.eicktech.com">WWW 。eicktech.com</a> ）。然而，微血管室的其他几个配置都是现成市售，与那些生活仪表系统（开发<a href="http://www.livingsys.com/">http://www.livingsys.com/</a> ）和IonOptix（ <a href="http://www.ionoptix.com/">http://www.ionoptix.com/</a> ）是很常见的。使用这些其他系统，它可能必须利用倒置显微镜，而不是一个传统的直立，虽然这是依赖于具体的实验和设备的要求。倒置显微镜的使用将是可取的实验protocOLS需要荧光或共聚焦成像。 </li><li>准备生理盐溶液（PSS的;所述），并确保pH值设置为特定的实验条件（7.38-7.42是典型的，属于生理范围内）到适当的水平。 PSS的解决方案，可事先准备和冷藏，但必须在使用前检查pH值适宜的温度。 PSS可以包含白蛋白，如果实验者或特定的协议要求，虽然这不是普遍强制性。 </li><li>应校准卡钳用来测量血管直径的数字视频，使用血球或显微镜阶段微米。精确的校准，这是至关重要的千分尺放置在血管腔的流入/流出微电极在同一平面上。 </li><li>附加微血管的移液器的领带回路应准备和易于后续使用。这些可以很容易地从8-0或小眼小号准备uture。单领带循环，可以事先准备，并存储在一个小的反转磁带，直到需要（这将保持他们丢失）带周围的显微镜幻灯片盖培养皿中。 （ 图3）。 </li></ol> 2。天实验<ol><li>应打开所有配套设备和适当的功能检查。这包括anti-vibration/floating表和循环恒温水浴（提供船只浴在适当的温度-常用37°C间），所有的压力传感器和船只室排水泵（ 图1A和1B）。打开船室和灌流灌流水库的平衡气体。 </li><li>填写所有的水库，管，商会与PSS的解决方案。灌流线，导致流入移液器必须填写完整，以避免气泡在这条线的存在，这可能驱逐和损坏的VAscular内皮细胞（ 图1C）。如果需要，使用注射器轻轻推动的PSS通过吸管，以确保它是完全充分的，并没有任何堵塞可能阻碍灌流流一路。流入压力应保持在合理的限度内，以避免损坏压力传感器。 </li></ol> 3。微血管收获<ol><li>是要采取微血管的动物从麻醉测定，船只问题，应根据所要研究的血管是最合适的程序分离。在某些情况下，这可能需要切除器官（如脑或冠状动脉微血管），而在其他国家，船只可以从麻醉动物（如肌肉）。例如船只在股薄肌的方向，提出了在图4。估计在体内的血管长度与前移走CEP证书或小卡钳。从动物/器官的血管被拆除之前，它可以是非常有益的，进行最后的检查，以确定该船只室准备和正常运作（包括全部到位领带循环）。 </li><li>从动物/机关抓船只的外观方面，在一端用细镊子，沿着船只的长度切割，直到它是免费的，格外小心，以避免或船只上的任何揪着拉船。一旦释放，立即将它充满与PSS在1.7 mL离心管，但不放手船只。这是要记住的方向，这样在洗澡的的灌流方向将是在动物的血液流动方向相同。 </li></ol> 4。插管的船只<ol><li>放置在充满浴船只和cannulate近端流入移液器。这样才能最好地完成套管通过了温和灌注率（〜50毫米汞柱）一双细镊子用来存放每一侧近端管腔墙（即每手钳子）​​。在我们的实验室中，我们用细镊子从精细的科学工具（ <a href="http://www.finescience.com/Home.aspx">http://www.finescience.com/Home.aspx</a> ），虽然任何适当的钳将有效地开展工作。滑的船只上的插管技巧和推进尖点，其中两个的领带循环可以确保船只上的船只。 </li><li>提高流入压力〜75毫米汞柱，进一步助长船只和促进远端枪头插管。流通过的船只，应被视为在该船只的远端流出（显微镜下）在灌流水库的失真。 Cannulate船只上流出吸管末端，并确保它有两个并列循环（这些最后的循环必须到位，插管前， 图5）。 </li><li>在XYZ坐标作为neede的调整，直到套管d，直到有失真最小的船只，船在体内的长度接近。提高流入的压力，直到它接近通常经历了由该船只段（我们实验室利用股薄肌8大阻力小动脉平均动脉压的80％）平均动脉压的百分比。 </li><li>提供适当的混合气体进入浴缸和地方的保鲜膜或玻璃盖在室内，以避免飞溅的显微镜镜头放置一个小气泡石。我们使用冒泡，通常是在所有宠物/水族店是直径约1厘米的石头，虽然规模较小，以及有效的工作。冒泡石的存在，确保适当的气体供应船只在任何时候都将延长船舶的可行性。在所有的测量周期（或空气流通过暂时中断的石头），以防止变形，这块石头被删除形象。释放流出吸管钳，并允许通过30分钟的船流。定期流动应夹紧在流出油管，以确定该船只正在开发休息音。在这一点上，所有船只必须检查压力泄漏。泄漏会引入到已知的船只（我们通常使用100-120毫米汞柱）的压力和夹紧流出，流入，线条明显。如果腔内的压力是稳定的，有没有明显的泄漏。然而，如果压力开始下降，一个重要的泄漏是目前必须关闭。流入或流出移液器泄漏一般可以通过增加一个额外的回路并列周围船只上的吸管纠正。另外，如果船只有一个小的分支，是允许的泄漏，这将危及船只的能力，有效地包含压力，可以引入一个额外的错误源。如果发现泄漏，它通常可以被关闭瓦特绑随着一个10-0眼科缝线的单回路。另外，从6-0缝合戏弄单链，使领带循环也是非常有效的。如果泄漏不能被识别，或可以不进行有效的捆绑关闭（例如，有没有的侧分支和简单地在血管壁上的洞主干），这可能在1实验过早终止和使用1替代成为船只必要（这通常是相同​​的船只在另一条腿 - 如有必要，或对侧 - 动物）。 船只应允许至少30分钟没有流量条件下平衡。所有的实验都无流量条件下进行。一旦船只已开发令人满意的音色和响应问题的实验纳入标准，该船是为后续研究做好准备。 这些入选标准可以根据不同的具体的实验方案和研究者。在我们的实验室中，我们经常UTilize了以下内容： <ol class="lalpha"><li> &gt; 30％（计算为ΔD/ D MAX×100，其中ΔD是平衡压力下的值在动脉内壁直径增加曝光的Ca 2 +无PSS和D MAX是最大的积极音直径测量中的Ca 2 +免费PSS的平衡压力）。 </li><li>生肌激活（维修，最好是减少，腔内压力增高的动脉直径）指数作为一个可行的血管平滑肌层。 </li><li>一个可行的内皮衬里，轻快的扩张对乙酰胆碱的反应（10 -6米的浴缸）。 </li><li>可以添加额外的入选标准，以适应具体的实验需要的协议，包括大蟒肾上腺素受体 ​​激动剂，如福林（10-7 M），扩张器，额外的刺激，如腺苷（10-5 </SUP&gt; M）或缺氧（O 2的平衡气体减少0％）。 </li></ol></li><li>下面的部分是“模拟实验”的例子。所有要求的解决方案是准备适当，注意与支付帐户的稀释因子在血管腔的PSS。作为一个例子，我们的一些设备，船只室容积为20毫升。正因为如此，20μL10 -2 M股票有效浓度在10-5中号船室洗澡解决方案的结果。实验是在去除血管，插管，平衡和验证如上所述，准备开始。如治疗随机和有效性的具体是在特定实验，并会消耗过多的空间，这方面的努力。因此，这些不包括细节。 初步控制反应的决心。举一个例子，如生肌激活（压力引起的收缩），intralumenal公关生理刺激是随机改变essure在所需的范围内，允许该船只适当的时间内响应（在这种情况下，我们将允许5-7分钟的时间内）。举一个例子，如与乙酰胆碱的挑战药理刺激，药物被添加到后面从库存解决方案，以随机顺序。在这种情况下，我们通常会使用范围从10-9到10-5米的浴缸，冲洗被定义为恢复平衡的直径。由于乙酰胆碱是一种快速反应剂，可以很容易地实现最大的扩张反应只有几秒钟。其他药物（如肽）可以花费更长的时间，引起极大的反应，这多少帐户，以及一段时间，采取有效冲刷。一旦控制的反应已收集，具体的干预措施可以对系统施加，包括船只孵化受体激动剂/拮抗剂，代理商在特定的离子通道行为，煽动改变腔内的压力，在针对特定的酶系统的平衡气体或药物制剂的差异，仅举几个最常见的干预。 干预后的适当时间是有效的（应适当测试），随后全面的数据收集可以启动。根据实验的具体协议，随后干预可以征收船只或第一，可以删除（通过冲洗 - 在可行的情况 - 或简单的生理挑战去除）。一旦船只已返回到其平衡状态（应核实），随后的干预措施，可应用于需要收集更多的数据。这是一般的程序将继续进行，直到实验终止或直到船只准备的质量低于先前确定的阈值标准（如响应激动剂或能力开发足够的活性音）。在这一点上，实验结束后，研究者可能希望收集的数据是独立的孤立船只的反应（如血管壁的力学，它可以进行使用生肌激活相同的过程，只是船只缺乏所有积极的基调14）。 </li><li>所有设备的定期清洗是必不可少的盐积聚的去除和防止任何有害生物实体的增长。在每个实验日结束，至少，完全刷新所有行和商会的PSS与蒸馏水或去离子水冲洗，并让其充分干燥。每2-3天使用后，用酒精洗净，以及执行和设备被允许完全干燥。在一贯使用的每个星期结束，所有线路和商会都装满0.1 M盐酸，允许以“孵化”30分钟，然后用清水洗净，充分如上。最后，在年底，连续使用2-3周更换新油管的所有部分，并与1.0 M盐酸清洗室和水库其次是一个广泛的蒸馏水/去离子水冲洗。如果有任何油管增长室/水库，这些被更换或适当彻底清洗油管或任何元素或视觉建立的变色。定期清洗和一般维修，设备可以持续很长一段时间。在写作时，我们的船室到8 日或9 年日常使用，我们经历了与他们没有显着的困难。 </li></ol> 5。代表结果<img alt="图1A" src="/files/ftp_upload/3674/3674fig1A.jpg" /> 图1A，这个数字呈现的基本的微血管站设置。一个=电视观看船只，B =数显卡尺，C =注射器推灌流THRough流入移液器必要，D =改变灌流流入压力的水压列，E =显微镜，F =微血管腔，G =灌流水库，H =压力显示器（可以根据需要添加多个）;我=微血管室排水泵为灌流; J =平衡气体混合物的坦克，K =废物中（灌流），L =循环热水器，M = anti-vibration/floating表。 <img alt="图1B" src="/files/ftp_upload/3674/3674fig1B.jpg" /> 图1B。这个数字提出了一个近距离观察的微血管室设置。这个数字：E =显微镜，F =微血管腔;高=压力监视器（当我们显示的简单只有一个，可以添加到多个不同地点，必要）;我=微血管室排水泵N =油管连接到一开始灌流流入移液器（图1A中的“D”的延续）;Ø=管路连接到灌流流出移液器的。 <p类“jove_content”&gt; <img alt="图1C" src="/files/ftp_upload/3674/3674fig1C.jpg" /> 图1C，这个数字呈现微血管室最近的观点。在这个数字中，n =灌流线（即，直接连接到灌流流入移液器管），O =管直接连接到灌流流出吸管;，P =流入移液器的水平和垂直位置控制，Q =灌流漏的水洗澡（图1A和1B连接&#39;我&#39;）; r =来自水库的水洗澡流入（连接到“G” 图1A）。 <img alt="图2" src="/files/ftp_upload/3674/3674fig2.jpg" /> 图2。这个数字提出了系统中的流体和气体的流动示意图。在此图中，= PSS灌流线流入，B = PSS灌流线流入滴管，C = PSS的灌流漏浴废物容器，D =<html> PSS的灌流液流出吸管流出，E =热水输入到室夹克，F =室夹克热水输出。 <img alt="图3" src="/files/ftp_upload/3674/3674fig3.jpg" /> 图3这个数字提出个人领带循环眼科缝合（通过解剖显微镜10X目镜和4.5X可调放大倍率看，A组），在我们准备使用的多个领带循环存储从8-0 10-0缝合扭转磁带上，在有盖培养皿中（以避免失去他们周围的显微镜幻灯片; B组）。 <img alt="图4" src="/files/ftp_upload/3674/3674fig4.jpg" /> 图4。这个数字提供了形象的股薄肌性动脉（通过一个标准的解剖显微镜观察）前手术的隔离和清除，允许适当的空间定位。 ve_content“&gt; <img alt="图5" src="/files/ftp_upload/3674/3674fig5.jpg" /> 图5。这个数字提供了一个空心微血管的形象代表。 A显示面板的整个长度，该船只的流入（A）和流出（二）移液器以及领带循环（c）所示。 B组高倍率图像，清楚地显示动脉内径数显卡尺（在这种情况下，直径为112微米）的决心。 <img alt="图6" src="/files/ftp_upload/3674/3674fig6.jpg" /> 图6。这个数字提出了一个空心的微血管以下平衡的代表图像。最上面的图片是控制的船只，无刺激，平衡压力的条件，中间的图像是同一船只扩张，挑战与乙酰胆碱（10 -6米的浴缸），和底部的图像，该船只是继constrictioN的挑战与福林（10-7米的浴缸）。 <img alt="图7" src="/files/ftp_upload/3674/3674fig7.jpg" /> 图7这个数字总结在响应一个孤立的微血管的挑战与回应：缺氧（面板，在我们的系统，减少在灌流灌流宝2〜135毫米汞柱〜40毫米汞柱），乙酰胆碱的浓度的增加（面板B）在血管内压力的控制条件下的变化和船只用无钙PSS的（C组）以下的孵化。

<ol>
	<li>Goodwill, A. G., Frisbee, S. J., Stapleton, P. A., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+Chronic+Anticholesterol+Therapy+on+Development+of+Microvascular+Rarefaction+in+the+Metabolic+Syndrome.">Impact of Chronic Anticholesterol Therapy on Development of Microvascular Rarefaction in the Metabolic Syndrome.</a> Microcirculation. , 1-18 (2009).</li><li>Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+vascular+thromboxane+generation+impairs+dilation+of+skeletal+muscle+arterioles+of+obese+Zucker+rats+with+reduced+oxygen+tension.">Increased vascular thromboxane generation impairs dilation of skeletal muscle arterioles of obese Zucker rats with reduced oxygen tension.</a> Am. J. Physiol. Heart Circ. Physiol. 295, H1522-H1528 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth-dependent+changes+in+endothelial+factors+regulating+arteriolar+tone.">Growth-dependent changes in endothelial factors regulating arteriolar tone.</a> Am. J. Physiol. Heart Circ. Physiol. 292, H207-H214 (2007).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogen+peroxide+emerges+as+a+regulator+of+tone+in+skeletal+muscle+arterioles+during+juvenile+growth.">Hydrogen peroxide emerges as a regulator of tone in skeletal muscle arterioles during juvenile growth.</a> Microcirculation. 15, 151-161 (2008).</li><li>Samora, J. B., Frisbee, J. C., Boegehold, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+myogenic+responsiveness+of+skeletal+muscle+arterioles+with+juvenile+growth.">Increased myogenic responsiveness of skeletal muscle arterioles with juvenile growth.</a> Am. J. Physiol. Heart Circ. Physiol. 294, 2344-2351 (2008).</li><li>Dacey, R. G., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+rat+intracerebral+arterioles:+methods,+morphology,+and+reactivity.">A study of rat intracerebral arterioles: methods, morphology, and reactivity.</a> Am. J. Physiol. Heart Circ. Physiol. 243, H598-H606 (1982).</li><li>Fredricks, K. T., Liu, Y., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Response+of+extraparenchymal+resistance+arteries+of+rat+skeletal+muscle+to+reduce+PO2.">Response of extraparenchymal resistance arteries of rat skeletal muscle to reduce PO2.</a> Am. J. Physiol. 267, H706-H715 (1994).</li><li>Durand, M. J., Raffai, G., Weinberg, B. D., Lombard, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiotensin-(1-7)+and+low-dose+angiotensin+II+infusion+reverse+salt-induced+endothelial+dysfunction+via+different+mechanisms+in+rat+middle+cerebral+arteries.">Angiotensin-(1-7) and low-dose angiotensin II infusion reverse salt-induced endothelial dysfunction via different mechanisms in rat middle cerebral arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 299, H1024-H1033 (2010).</li><li>LeBlanc, A. J., Cumpston, J. L., Chen, B. T., Frazer, D., Castranova, V., Nurkiewicz, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+inhalation+impairs+endothelium-dependent+vasodilation+in+subepicardial+arterioles.">Nanoparticle inhalation impairs endothelium-dependent vasodilation in subepicardial arterioles.</a> J. Toxicol. Environ. Health A. 72, 1576-1584 (2009).</li><li>Jernigan, N. L., LaMarca, B., Speed, J., Galmiche, L., Granger, J. P., Drummond, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dietary+salt+enhances+benzamil-sensitive+component+of+myogenic+constriction+in+mesenteric+arteries.">Dietary salt enhances benzamil-sensitive component of myogenic constriction in mesenteric arteries.</a> Am. J. Physiol. Heart Circ. Physiol. 294, H409-H420 (2008).</li><li>Stapleton, P. A., Goodwill, A. G., James, M. E., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mechanisms+of+endothelium-dependent+dilation+in+skeletal+muscle+arterioles+with+genetic+hypercholesterolemia.">Altered mechanisms of endothelium-dependent dilation in skeletal muscle arterioles with genetic hypercholesterolemia.</a> Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1110-R1119 (2007).</li><li>Goodwill, A. G., Stapleton, P. A., James, M. E., d'Audiffret, A. C., Frisbee, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+studying+isolated+resistance+vessel+from+rabbit+mesentery+and+brain+and+their+responses+to+drugs.">A method for studying isolated resistance vessel from rabbit mesentery and brain and their responses to drugs.</a> Circ. Res. 4, 525-536 (1967).</li><li>Davis, M. J., Kuo, L., Chilian, W. M., Muller, J. M. I. s. o. l. a. t. e. d., Barker, J. H., Anderson, G. L., Menger, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+23.">Chapter 23.</a> Isolated, perfused microvessels. In: Clinically Applied Microcirculation Research. 32, 435-456 (1995).</li><li>Lombard, J. H., Liu, Y., Fredricks, K. T., Bizub, D. M., Roman, R. J., Rusch, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

没有利益冲突的声明。

提出的协议描述的骨骼肌微血管隔离，清除和双插管，虽然这一般技术可以很容易地适用于大多数组织。对于当前的手稿，“动脉”一词已被用于由作者来形容一个阻力血管直径在70-120微米休息积极的基调下，这也是一个主要因素调节器官灌注阻力或之间不等组织。 一些修改，这个系统可以安装多个应用程序的具体调查，并随时可以提供更深入的实验（例如，17膜电?...

<table border="1"><tbody><tr><td> 试剂和设备 </td><td> 公司 </td><td> 评论/目录＃ </td></tr><tr><td>船厅</td><td>习俗</td><td>戴夫Eick（都城） </td></tr><tr><td>加热循环水浴</td><td> PolyScience和哈克</td><td>哈克直流10 </td></tr><tr><td>滴管</td><td>冯检基Haer公司</td><td>毛细管2.0 mm外径×1.0 mm内径（27-33-1） </td></tr><tr><td>血压计</td><td>世界精密仪器</td><td></td></tr><tr><td>水夹套水库的</td><td>习俗</td><td></td></tr><tr><td>外部光源</td><td>世界精密仪器</td><td> NovaFlex经股</td></tr><tr><td>吸取普勒</td><td>微观仪器</td><td> PMP102 Micropipet普勒</td></tr><tr><td>全部完井MENT的手术工具</td><td>罚款科学工具</td><td>杜蒙</td></tr><tr><td>超精细镊子</td><td>罚款科学工具</td><td> INOX＃5 </td></tr><tr><td>丝绸缝合线</td><td> ethilon </td><td> ＃10-0或9-0 </td></tr><tr><td>立体显微镜</td><td>奥林巴斯</td><td>奥林巴斯SZ-11 </td></tr><tr><td>模拟视频卡钳</td><td> boeckeler </td><td>通过控制器（VIA-100） </td></tr><tr><td>高分辨率模拟摄像机</td><td>松下</td><td> GP - MF 602 </td></tr><tr><td>氧气罐</td><td>区域性</td><td>余额21％的氮和5％CO 2的平衡氮</td></tr><tr><td>管道</td><td>聚乙烯</td><td></td></tr><tr><td>排水泵</td><td>科尔帕默仪器有限公司</td><td></td></tr><tr><td>修饰大鼠的PSS </td><td>看到配方如下</td><td></TD&gt; </tr><tr><td>凡Breemen松弛的PSS </td><td>看到配方如下</td><td></td></tr></tbody></table> 表1。隔离的微血管站提出的数字设置的主要组成部分的清单。 <table border="1"><tbody><tr><td> 修改大鼠的PSS食谱 </td><td> 为了使两公升的PSS </td><td> 20X盐库存（2L） </td><td> 20X缓冲库存（2L） </td></tr><tr><td>氯化钠</td><td></td><td> 278.0Ğ </td><td></td></tr><tr><td>氯化钾</td><td></td><td> 14.0Ğ </td><td></td></tr><tr><td>硫酸镁4-7H 2 O的 </td><td></td><td> 11.5Ğ </td><td></td></tr><tr><td>氯化钙2-H 2 O </td><td></td><td> 9.4Ğ </td><td></td></tr><tr> &lt;TD&gt;碳酸氢钠3 </td><td></td><td></td><td> 80.8Ğ </td></tr><tr><td> EDTA的</td><td></td><td></td><td> 0.4Ğ </td></tr><tr><td>的NaH 2 PO 4计 </td><td> 0.28Ğ </td><td></td><td></td></tr><tr><td>葡萄糖</td><td> 1.98Ğ </td><td></td><td></td></tr><tr><td> 20倍的盐库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td> 20X缓冲库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td>蒸馏水</td><td> 1800毫升</td><td></td><td></td></tr></tbody></table> 表2。配方为标准生理盐溶液中用于孤立微血管协议“（PSS）。 食谱上的评论：2公升盐库存和缓冲库存的大号2。这些C冷藏不使用时，但动摇他们之前往往准备的PSS。额外的成分，添加在最终的PSS的准备时间。 <table border="1"><tbody><tr><td> 凡Breemen松弛的PSS </td><td> 为了使2升的PSS </td><td> 20X盐库存（1L） </td><td> 20X缓冲库存（1L） </td></tr><tr><td>氯化钠</td><td></td><td> 107.4Ğ </td><td></td></tr><tr><td>氯化钾</td><td></td><td> 7.0Ğ </td><td></td></tr><tr><td>硫酸镁4-7H 2 O的 </td><td></td><td> 5.76Ğ </td><td></td></tr><tr><td>氯化镁2-6H 2 </td><td></td><td> 81.32Ğ </td><td></td></tr><tr><td> 碳酸氢钠 </td><td></td><td></td><td> 40.4Ğ </td></tr><tr><td>美东时间一</td><td></td><td></td><td> 0.2Ğ </td></tr><tr><td> EGTA </td><td></td><td></td><td> 15.22 </td></tr><tr><td>的NaH 2 PO 4计 </td><td> 0.28Ğ </td><td></td><td></td></tr><tr><td>葡萄糖</td><td> 1.98Ğ </td><td></td><td></td></tr><tr><td> 20倍的盐库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td> 20X缓冲库存</td><td> 100毫升</td><td></td><td></td></tr><tr><td>蒸馏水</td><td> 1800毫升</td><td></td><td></td></tr></tbody></table> 表3。配方范Breemen松弛生理盐溶液（PSS），在孤立的微血管协议的零积极音的条件下使用。 食谱上的评论：1 L一盐库存ND 1升的缓冲库存。不使用时，可以冷藏，但动摇他们之前往往准备的PSS。额外的成分，添加在最后松弛的PSS的准备时间。

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

隔离的微血管准备是体外制备，允许考试因素的控制血管直径的不同贡献，因此，灌注阻力1-5。这是一个经典的实验准备，在很大程度上，最初是由内田等 15，几十年以前。这最初的描述提供了进行了广泛的修改和提高，主要是在实验室的布赖恩博士杜陵在弗吉尼亚6-8大学，技术的基础上，我们提出了电流的方法，在下面的网页。这种准备，具体是指在大鼠作为首选微血管纤细的动脉，但基本的准备，可以很容易地应用到几乎任何其他组织或器官9-13跨物种隔离的船只。机械（即二维）在孤立的微血管的变化，可以很容易地评估在浩如烟海的生理（例如，缺氧，血管内的压力，或剪切）或药理的挑战，并能提供洞察到机械元素组成一个完整的综合措施，虽然体外 ，组织响应。这种方法的意义是，它允许简便操作的影响微血管直径的综合调控，同时也让许多从其他来源，包括血管内压力（生肌），自主支配，血流动力学的贡献控制（例如，剪切应力），内皮依赖或独立的刺激，激素，实质影响，提供了部分清单。在适当的实验条件和适当的目标，这可以作为一个优势在体内或原位组织/器官的筹备工作，不轻易让浅显的控制更广泛的系统性变量。 马约旦基本上是该制剂的限制其优势的结果。根据定义，这些船只的行为被许多最重要的贡献者调节血管阻力已被删除，包括神经，体液免疫，代谢，因此等的条件下，研究，研究者应注意避免过度所收集的数据利用这个准备的解释和推断。该制剂方面关心的其他重要领域，它可以很容易损坏，如内皮衬里或血管平滑肌细胞成分，如变量的错误源可以被引入。强烈建议，个别研究者利用，以确保该制剂的质量，无论是在实验开始，并定期在整个协议的过程中适当的测量。

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

一个<em&gt;体外</em&gt;编制说明两个血管反应性血管活性物质的刺激和通过被动墙力学的基本结构性能评估的审讯股薄肌的最大阻力小动脉的隔离。

“体外骨骼肌微血管制备离体血管反应性的调查

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

the-ex-vivo-isolated-skeletal-microvessel-preparation-for

Research

JoVE Journal

Biology

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

10.6K 次观看.  West Virginia University. 一个体外编制说明两个血管反应性血管活性物质的刺激和通过被动墙力学的基本结构性能评估的审讯股薄肌的最大阻力小动脉的隔离。

视频: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

在体内微循环在骨骼肌内重要的显微镜测量

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

科研

生物学

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

对离体乳头肌的心脏收缩力对小鼠的研究收缩尺寸

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

在c-fos蛋白免疫组织学检测：一个有用​​的工具，中央通路在特定的生理反应所涉及的标记<em&gt;在体内</em&gt;和<em&gt;离体</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

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.