Name: en face preparation of mouse blood vessels
Uploaded: 2017-05-19T13:00:00.000+00:00
Duration: 10 min 1 s
Description: Watch this Scientific Journal Video about En Face Preparation of Mouse Blood Vessels at JoVE.com

作者的研究活动得到了国家卫生研究院对Abe博士（HL-130193，HL-123346，HL-118462，HL-108551）的资助。

小鼠部分颈动脉结扎和分离小鼠主动脉和颈动脉用于面部免疫染色的方案由机构动物护理和使用委员会（IBT 2014-9231）批准。 左侧部分颈动脉结扎<ol><li>通过在桌子上放置一个12英寸x 14英寸的加热垫来准备手术空间，并用大的干净的手术罩盖住垫子和桌面。调整吊架的臂，使立体显微镜的视野位于加热垫的中心区域。 </li><li> 打开桌面上的加热垫，并将3设置控制拨盘设置为中等热位。在此温度设置下，外科手术板表面（见1.6.1）将为38-40°C。 <ol><li>将一个干净的笼子放在另一个加热垫上。如上所述打开加热垫。这种笼子将用于手术后的恢复（见1.16）以及房屋。 </li></ol></l我&gt; <li>在手术台上放置包含虹膜剪刀（1对），组织钳（1对），超级夹钳（2对），弹簧剪刀（1对），钝头牵开器（1对; 2.5 mm宽）的高压灭菌消毒袋， （1），消毒6-0丝线缝合线，棉花贴片机，迷你棉花贴纸机，手术用纱和2"×2"纱布海绵。还放置一个含有70％乙醇的挤压瓶，另一个装有洗必太洗手液的外科手术台。 </li><li>称重一只老鼠需要体重来确定适当的镇痛量，这将在手术前立即进行。 </li><li>将鼠标放入感应室。 </li><li> 打开氧气罐和麻醉剂蒸发器，以便在感应室中麻醉鼠标。维持2％的异氟烷​​水平。鼠标停止移动前需要3-5分钟。 <ol><li>鼠标正在麻醉时在立体显微镜下放置一小块无菌手术罩（24英寸×24英寸）以创建手术表面。然后将丙烯酸手术板（已用70％酒精清洗）放在悬垂的表面上。因此，手术板应该在加热垫上，但是由两层手术包装隔开。 </li></ol></li><li>当鼠标停止在感应室中移动时，将鼠标移动到手术前准备区域，并将其鼻子定位到与蒸发器（2％异氟烷）连接的鼻锥中。使用电动修剪器或脱毛剂洗剂去除颈部周围的头发。我们推荐脱毛乳液，因为这种方法不会产生松散的头发片，难以从外科手术区域彻底清除。 </li><li> 将鼻锥放在适当的位置，将鼠标移动到手术板上。 <ol><li>将左右前爪向下拉到手术板。将两腿后腰缠住在鼠标的右侧。这导致鼠标体的轻微旋转，使得鼠标的颈部区域的左侧变得更好地定位用于手术。 </li></ol></li><li>用70％酒精，氯己定手术擦洗，再用70％酒精消毒切口区域。用除了颈部切口区域的灭菌手术盖子盖住鼠标。 </li><li>通过脚趾确认通过腹膜内或皮下注射完全麻醉并给予镇痛（卡洛芬3-5mg / kg）。 </li><li>在解剖显微镜下，使用手术刀或虹膜剪刀在颈部周围进行腹侧中线切口。 注意:我们使用剪刀，因为立体显微镜的工作距离是有限的，这使得难以使用手术刀。 </li><li>将左颈总动脉（LCCA）推开，将覆盖血管的唾液腺重新放置在左侧nimal。 </li><li>识别手术领域的所有血管（ 图1 ）。 LCCA分叉到左内颈动脉（ICA）和左外颈动脉（ECA）。浅层甲状腺动脉（STA）来自内侧ECA。枕叶动脉（OA）通常来自ECA，但在一些小鼠中，它起源于ICA。 </li><li> 使用灭菌的6-0丝线缝合除OA外的所有动脉分支。要做到这一点，做以下两个连接。 <ol><li>轻轻取出左颈内动脉（ICA）周围和下方的结缔组织。用镊子抓住一条预切线6-0丝缝合（约2.5厘米），并将其通过动脉。使用另一对镊子，将缝合线拉长约1/3的长度并连接动脉。 </li><li>以与上述相同的方式去除左外颈动脉（ECA）周围的结缔组织，并在左上甲状腺上进行结扎d动脉（STA）（ 图1 ）。注意不要损伤外科手术领域的神经纤维。 </li></ol></li><li>当进行这些连接时，将唾液腺返回原来的位置，并放置2-3滴无菌盐水使手术区水合。使用6-0涂层的维克罗缝线缝合皮肤。 </li><li>手术后，将鼠标置于预热笼内（见1.2.1）。鼠标应该在5分钟内醒来，开始走动。一旦确认鼠标的行为正常，将笼子带到动物房间。 </li><li>在恢复前3天每天观察小鼠。只要实验要求在实验协议要求的各种条件下，鼠标就可以保持。可以在手术后的任何时间进行面部准备。 </li></ol> 面部免疫染色<ol><li>通过吸入过量安乐死小鼠CO 2 。 </li><li>将鼠标放在仰卧位（腹侧向上）位置到解剖板上。 </li><li>通过使用虹膜剪刀进行中线切口暴露腹腔。 </li><li>通过将胸骨横向切割到胸骨来暴露胸腔。 </li><li>在腔静脉中切口或切断股动脉以排出血液。 </li><li>将插入重力灌注装置（120厘米水压）的26 G针插入左心室的顶点，并用含有肝素（40 U / mL）的盐水溶液灌注循环系统。继续灌注，直到从切口中流出的盐水变得清晰。 </li><li>将灌注系统从生理盐水切换到PBS（磷酸盐缓冲盐水）中含有4％多聚甲醛的固定溶液，并继续灌注5分钟。 </li><li>使用平头剪刀和镊子收获主动脉和左颈动脉和右颈动脉，并置于含有固定在冰上的50mL锥形管中。 </li><li&gt;在解剖显微镜下将血管转移到含有PBS的培养皿中，小心地取出附着于主动脉和颈动脉的脂肪和结缔组织。将主动脉和颈动脉纵向分离并分离以暴露内皮（ 图2 ）。 </li><li>将每个容器分别转移到含有0.5mL渗透溶液（PBS中0.1％Triton X-100）的12孔板的孔中。在室温（RT）摇动下使血管渗透10分钟。 </li><li>用PBS短暂洗涤。 </li><li>为了阻断非特异性抗体结合位点，在TTBS（含有2.5％吐温20的Tris缓冲盐水（TBS））中，将来自已经制备了二次抗体的动物物种的10％正常血清中的血管孵育30分钟，在RT摇摆。 </li><li>孵育具有在10％正常血清（如上所述）的TTBS中适当稀释的初级抗体的血管在4℃下摇摆过夜。水平必须确定每种抗体的稀释度。 </li><li> 进行以下对照染色。 <ol><li>用TTBS而不是一级抗体孵育血管，然后与第二抗体一起孵育。 </li><li>孵育含TTBS的血管，其中含有一级抗体的相同动物（物种）的非免疫（或预免疫）血清或Ig，然后与第二抗体一起孵育。 </li><li>省略与二级抗体孵育。必须以相同的方式处理这些对照样品，同时进行用特异性抗体染色的同时。 </li></ol></li><li>用TTBS洗涤血管3次，每次10分钟，在室内摇摆。 </li><li>用在10％正常血清（如上所述）的TTBS中适当稀释的荧光标记的二次抗体孵育1小时，在室温下摇动。可以同时进行DAPI（4&#39;，6-二脒基-2-苯基吲哚）的核染色通过在H 2 O中加入1 / 5,000体积的含有5mg / mL DAPI的DAPI储备溶液。 </li><li>用TTBS洗涤3次，每次10分钟，摇匀。 </li><li>在PBS中短暂冲洗。 </li><li>将一滴防褪色试剂放在玻璃罩（22 mm x 50 mm）上，并将血管置于玻璃上，使内皮朝下。 </li><li>在血管上放置载玻片（22 mm x 75 mm），同时避免陷入气泡。 </li><li>将幻灯片放在干净的实验室擦拭物（ 例如 Kimwipe）上，并用两块实验室擦拭物盖住幻灯片。在载玻片上轻轻放置3.5公斤重量（ 例如使用一瓶水放在厚厚的书上），最多5分钟，以使平面血管样本平坦化。 </li><li>取出重量，擦去盖玻片周围的多余溶液。 </li><li>在盖玻片的四个角落涂抹指甲油，将幻灯片放在幻灯片中，盖玻片朝上，并保持在黑暗中（或4℃）过夜。该过程使组织进一步变平，并使得更容易以高放大倍率进行显微镜检查。 </li><li>使用指甲油完全密封盖玻片。 </li><li>指甲油干燥后立即进行显微镜检查。 </li><li>如果需要，将载玻片存放在-20°C。 </li></ol>

<ol>
	<li>Jelev, L., Surchev, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+simple+technique+for+en+face+endothelial+observations+using+water-soluble+media+-'thinned-wall'+preparations.">A novel simple technique for en face endothelial observations using water-soluble media -'thinned-wall' preparations.</a> J Anat. 212 (2), 192-197 (2008).</li><li>Neill, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+on+venous+endothelium+of+alterations+in+blood+flow+through+the+vessels+in+vein+walls,+and+the+possible+relation+to+thrombosis.">The effect on venous endothelium of alterations in blood flow through the vessels in vein walls, and the possible relation to thrombosis.</a> Ann Surg. 126 (3), 270-288 (1947).</li><li>Rogers, K. A., Kalnins, V. I. <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+examining+the+endothelial+cytoskeleton+in+situ+using+immunofluorescence.">A method for examining the endothelial cytoskeleton in situ using immunofluorescence.</a> J Histochem Cytochem. 11 (11), 1317-1320 (1983).</li><li>Poole, J. C. F., Sanders, A. G., Florey, H. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regeneration+of+aortic+endothelium.">The regeneration of aortic endothelium.</a> J Pathol Bacteriol. 75, 133-143 (1958).</li><li>Sade, R. M., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=En+face+stripping+of+vascular+endothelium.">En face stripping of vascular endothelium.</a> Microvasc Res. 4, 77-80 (1972).</li><li>White, G. E., Gimbrone, M. A., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+influencing+the+expression+of+stress+fibers+in+vascular+endothelial+cells+in+situ.">Factors influencing the expression of stress fibers in vascular endothelial cells in situ.</a> J Cell Biol. 97 (2), 416-424 (1983).</li><li>Kim, D. W., Gotlieb, A. I., Langille, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+modulation+of+endothelial+F-actin+microfilaments+by+experimental+alterations+in+shear+stress.">In vivo modulation of endothelial F-actin microfilaments by experimental alterations in shear stress.</a> Arteriosclero. 9, 439-445 (1989).</li><li>Haka, A., Potteaux, S., Fraser, H., Randolph, G., Maxfield, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+monocyte+subpopulations+in+murine+atherosclerotic+plaques+by+multiphoton+microscopy.">Quantitative analysis of monocyte subpopulations in murine atherosclerotic plaques by multiphoton microscopy.</a> Plos ONE. 7 (9), 244823e (2012).</li><li>Ch&#232;vre, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Resolution+imaging+of+intravascular+atherogenic+inflammation+in+live+mice.">High-Resolution imaging of intravascular atherogenic inflammation in live mice.</a> Circ Res. 114, 770-779 (2014).</li><li>Heo, K. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disturbed+flow-activated+p90RSK+kinase+accelerates+atherosclerosis+by+inhibiting+SENP2+function.">Disturbed flow-activated p90RSK kinase accelerates atherosclerosis by inhibiting SENP2 function.</a> J Clin Invest. 125 (3), 1299-1310 (2015).</li><li>Le, N. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+crucial+role+for+p90RSK-mediated+reduction+of+ERK5+transcriptional+activity+in+endothelial+dysfunction+and+atherosclerosis.">A crucial role for p90RSK-mediated reduction of ERK5 transcriptional activity in endothelial dysfunction and atherosclerosis.</a> Circ. 127, 486-499 (2013).</li><li>Kano, Y., Katoh, K., Masuda, M., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+composition+of+stress+fiber-plasma+membrane+attachment+sites+in+endothelial+cells+in+situ.">Macromolecular composition of stress fiber-plasma membrane attachment sites in endothelial cells in situ.</a> Circ Res. 79, 1000-1006 (1996).</li><li>Nigro, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclophilin+A+is+an+inflammatory+mediator+that+promotes+atherosclerosis+in+apolipoprotein+E-dependent+mice.">Cyclophilin A is an inflammatory mediator that promotes atherosclerosis in apolipoprotein E-dependent mice.</a> J Exp Med. 208 (1), 53-66 (2011).</li><li>Mattson, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+arterial+blood+pressure+in+different+strains+of+mice.">Comparison of arterial blood pressure in different strains of mice.</a> Am J Hypertens. 14 (5), 405-408 (2001).</li><li>Jinguji, Y., Fujiwara, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+fiber+dependent+axial+organization+of+fibronectin+fibrils+in+the+basal+lamina+of+the+chick+and+mesenteric+artery.">Stress fiber dependent axial organization of fibronectin fibrils in the basal lamina of the chick and mesenteric artery.</a> Endothelium. 2, 35-47 (1994).</li><li>Neckel, P. H., Mattheus, U., Hirt, B., Lust, L., Mack, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+tissue+clearing+(PACT):+Technical+evaluations+and+new+perspectives+in+immunofluorescence,+histology,+and+ultrastructure.">Large-scale tissue clearing (PACT): Technical evaluations and new perspectives in immunofluorescence, histology, and ultrastructure.</a> Sci Rep. 6, 34331 (2016).</li></ol>

当处理小鼠血管时，重要的是记住内皮是脆弱的，并且任何过量的机械力将损伤内皮细胞。例如，如果容器被强力灌注，则血管壁破裂或分离，当使用手动注射器灌注脉管系统时，这可能容易发生。 为了获得恒定的灌注压力，我们使用120厘米水柱压力的重力灌注系统。据报道，不同于应变的小鼠的平均动脉压在130和170厘米H 2 O 14之间 。因此，我们使用的灌注压力略小于测得的动脉压。当我们灌注固定的大鼠主动脉时，使用90cm H 2 O柱压力6 。 如果容器在收获期间被拉伸，原位内皮细胞也会受损，清洁，纵向分割，免疫染色和安装。事实上，机械损伤是在面部制剂中失去内皮细胞的常见原因之一。容器拉伸可以在程序中的任何步骤发生，但最常见的是在收集容器时发生。除去附着于外膜的脂肪组织时，容易拉伸血管。 通过沿其整个长度纵向切割血管来进行面部准备。这通常使用尖锐的眼科剪刀。然而，如果目标血管的内径小，则剪刀的尖端可能太大。在这种情况下，可以使用断裂的薄的一次性剃刀刀片进行切割。我们使用这种技术来制作肠系膜动脉15 。 固定后，面部准备渗透。通常，使用含有Triton X-100的PBS但是可以使用其他透化试剂如Tween-20，Nonidet P-40，皂苷，皂角苷和Leucomerm。理想情况下，每个实验室应优化透化条件。对于小鼠主动脉和颈动脉，我们在室温下用含有0.1％Triton X-100的PBS处理10分钟，并且该处理足以透化血管壁内的所有细胞。然后首先用一级抗体依次处理渗透性样品，然后再进行荧光标记的二抗。对于染色小鼠血管，至关重要的是，一抗不是由小鼠制成的，因为小鼠血管组织将含有将被荧光标记的次级抗小鼠IgG标记的小鼠IgG，导致高背景染色。对于显微镜，样品应尽可能平坦。我们用3.5公斤的重量压载玻片5分钟。根据经验确定该比重。 w ^通过使用普通的荧光显微镜，激光扫描共聚焦显微镜和多光子显微镜，可以将面部制剂标记为免疫荧光。共聚焦显微镜最好从血管表面获得高达50μm左右的内皮和内皮下区域的图像，而通过多光子照明模式实现的激发光的深度穿透使得能够从深度距血管壁表面最远2mm。此外，多光子显微镜可用于二次谐波成像，最通常用于研究血管壁中的胶原纤维组织。面对准备很大，允许我们调查大血管区域，如主动脉的整个长度。这些是使用免疫荧光染色的面部血管制剂的一些优点。然而，这种技术有一些缺点。首先，方法只限于特异性抗体和其他荧光标记试剂如荧光鬼笔环肽，DAPI和DiI-Ac-LDL（由1,1&#39;-二十八烷基-3,3,3&#39;，3&#39;-四甲基 - 吲哚羰花青标记的乙酰化低密度脂蛋白高氯酸盐）。由于成像是基于荧光，因此不能对样本的非荧光部分进行成像。整个安装标本通常表现出自发荧光，而在大血管中发现的弹性蛋白纤维发绿色。当使用落射荧光显微镜时，这种荧光是特别有问题的;因此，强烈建议通过共焦显微镜进行成像。然而，通过使用用红色荧光染料标记的二次抗体，可以显着降低该绿色自发荧光的干扰。最后，由于面部样本很厚，透射照明的成像是不可能的。 商业共焦和多光子显微镜附带图像分析软件一个用于定量分析图像的荧光强度。如果在同一张幻灯片中进行比较，量化数据更可靠。这是因为所有的样品免疫染色条件都是一样的。如果不同载玻片之间的染色强度必须进行比较（例如，正常与病变的血管），则验证数据的唯一方法是增加样本数，从而可以平均技术和生物学变化。通常，在容器表面附近获得的共焦图像上的强度测量更可靠。由组织的更深的区域获得的图像的荧光强度倾向于变化更大，因为激发和发射的荧光的散射和吸收的程度不同。一般来说，必须小心解释在深层区域检测到的微小的强度差异。为了提高来自组织较深区域的成像能力，正在尝试使t问题半透明，并且获得了令人印象深刻的图像（例如，见Neckel 等人最近的一篇文章16和这些作者引用的论文）。尽管用于使组织半透明的处理可能会提取某些抗原和/或使某些表位变性，但是该方法可用于从较深的组织区域获得更多可重现的荧光信号。 使用面部制剂不仅限于通过荧光显微镜进行成像。使用立体显微镜，可以使用面容器制剂来研究用油红O染色后的动脉粥样硬化斑块形成的程度。可以无菌制备面容器制剂。这样的制剂可以保持在培养物中，并且可以用作离体系统来研究白细胞 - 内皮细胞相互作用。

通过光学显微镜进行组织病理学研究，常规处理生物组织三维片段进行石蜡包埋，然后进行切片和染色。石蜡包埋的组织样品可以在所有三维中为几毫米。然而，为了光学显微镜的目的，它必须首先切片，使得光可以通过然后染色，使得薄部分产生足够的成像对比度。由于切片样品的厚度通常为5-10微米，所以每次只能看到整个样品的二分之一。可以收集连续的部分，并且在单独成像每个部分之后，对3D图像进行计算机辅助重建，但是这是一个繁琐的工作。血管的组织病理学，特别是研究动脉粥样硬化的发病机制，具有独特的问题。动脉粥样硬化是一种发展的重点疾病局部发生血流紊乱的地区。此外，该疾病是在内膜内开始的，一个由大量动脉的内皮细胞和细胞外基质的单层组成的薄组织。由于这些原因，使用分段血管来定位和研究早期病变是一个挑战，因为可以容易地错过切片病变。即使部分确实包括患病区域，在介质和外膜中只能看到含有内皮细胞和其他血管壁细胞的5-10μm部分。 整个安装面（发音为änfäs）的准备工作使我们可以从主动脉根部一直到髂总动脉，调查血管表面的广泛区域，如整个主动脉。使用这样的标本用特异性抗体和其他特异性探针染色，可以确定病变的位置，同时也可以在内皮细胞中发生各种分子事件动脉粥样硬化形成，如蛋白质的表达，定位和翻译后修饰的变化。除了研究动脉粥样化形成外，在面部制剂中观察到的内皮细胞形状被用作区域时间平均血流模式的指标。这些数据对于研究原位内皮细胞的机械信号是很重要的。为此，常规的组织学横切血管是无用的。因此，对于血管医学和生物学，特别重要的是获得一种用于制造血管的面部制备技术，其允许观察血管表面的广泛区域以及血管的较深的地下区域。 正如Jelev和Surchev 1所述 ，血管生物学家已经开发了各种观察血管内衬的方法。一些巧妙的方法是在20世纪40年代和50年代开发的。他们使用这些方法<html>能够研究排列血管内表面的内皮细胞的基本组织。然而，由于这些准备工作的准备方法（所谓的Hautchen方法2,3,4或容器表面5的剥离）以及样品染色的方式，并不总是可以获得不间断的形态信息从血管表面进入血管壁的较深的区域。全面安装和面部血管制剂联合免疫荧光染色使我们不仅可以研究这些细胞内皮细胞的形态和蛋白质表达和定位，而且还将这些研究扩展到血管壁的内皮下区域。早期研究使用血管，面部免疫荧光染色剂的制剂在20世纪80年代开始出现6 ，随着激光扫描共聚焦显微镜和最近的多光子显微镜的出现，现在可以获得免疫荧光染色的面容器样品中的血管壁结构的清晰的对焦图像以及活体动物中的血管网络这些基于计算机的成像技术创建了对焦光学分割图像，并且通过堆叠这样的图像，可以获得组织中血管壁和血管网络的重建3D图像，另外，一个可以生成沿着重建图像12,13的Z轴形成的部分的图像。 在本文中，我们将说明一种制备小鼠主动脉和颈动脉免疫荧光染色的方法。面对准备即使在这些容器被实验操作之后也可以制造。例如，可以部分地连接颈动脉，然后在这样的手术之后进行面部制备。因此，我们还将在本文中描述我们如何在颈动脉上进行部分结扎。与大型动物如大鼠，兔子和人类进行类似的制备相比，小鼠血管的尺寸较小且更易碎，因此需要对外科手术分离血管进行处理以及进行抗体染色和显微镜检查。因为最常用的遗传修饰动物模型是老鼠，因此许多研究者在不伤害它们的情况下处理小鼠血管变得至关重要。在本手稿中，我们将介绍如何在小鼠主动脉和颈动脉进行面部准备时处理小鼠血管。为了展示的目的，我们将使用野生型C57 / b6小鼠。

<table><tbody><tr><td>0.9% Sodium Chloride injection solution USP 500 mL bag</td><td>Fisher Scientific</td><td>NC9788429</td><td></td></tr><tr><td>12-well plates</td><td>Fisher Scientific</td><td>12556005</td><td></td></tr><tr><td>6-0 coated vicryl suture</td><td>Ethicon</td><td>J833G</td><td></td></tr><tr><td>AF488 goat anti-rat IgG&amp;nbsp;</td><td>Life Technologies</td><td>A11006</td><td></td></tr><tr><td>AF546 goat anti-rabbit IgG&amp;nbsp;</td><td>Life Technologies</td><td>A11035</td><td></td></tr><tr><td>Anti-CD144 (Ve-Cad)</td><td>BD Biosciences</td><td>BD555289</td><td></td></tr><tr><td>Anti-VCAM-1 (H-276) Rabbit polyclonal IgG</td><td>Santa Cruze Biotechnology</td><td>Sc-8304</td><td></td></tr><tr><td>Aoto Flow System</td><td>Braintree Scientific</td><td>EZ-AF9000</td><td></td></tr><tr><td>Autoclave Wrap. 24 inch x 24 inch</td><td>Cardinal Health</td><td>4024</td><td></td></tr><tr><td>Blunt retractors, 2.5 mm wide</td><td>Fine Science Tools</td><td>18200-10</td><td></td></tr><tr><td>Caprofen (Rimadyl)&amp;nbsp;</td><td>zoetis</td><td>NADA#141-199</td><td></td></tr><tr><td>Chlorhexidine Scrub, 2%</td><td>Med-Vet International</td><td>RXCHLOR2-PC</td><td></td></tr><tr><td>Curity gauze sponges 2 x 2</td><td>Cardinal Health</td><td>KC2146</td><td></td></tr><tr><td>Electric heating pad, 12 x 14</td><td>Fisher Scientific</td><td>NC0667724</td><td></td></tr><tr><td>Extra Fine Graefe Forceps</td><td>Fine Science Tools</td><td>11152-10</td><td></td></tr><tr><td>Iris Scissors</td><td>Fine Science Tools</td><td>14090-11</td><td></td></tr><tr><td>Micro cover glass 22 mm x 50 mm</td><td>VWR</td><td>48393059</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-18</td><td></td></tr><tr><td>normal goat serum</td><td>Equitech-Bio</td><td>GS05</td><td></td></tr><tr><td>Paraformaldehyde Solution 4% in PBS</td><td>Santa Cruze Biotechnology</td><td>SC-281692</td><td></td></tr><tr><td>Petri dishes 100 mm x 15 mm</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>Prolong Gold Antifade mountant with DAPI</td><td>Life Technologies</td><td>P-36935</td><td></td></tr><tr><td>Puritan cortton swabs</td><td>VWR</td><td>10806-005</td><td></td></tr><tr><td>Puritan Mini cotton tipped aplicators</td><td>VWR</td><td>82004-050</td><td></td></tr><tr><td>Round handled Needle Holder</td><td>Fine Science Tools</td><td>12076-12</td><td></td></tr><tr><td>Silk Suture 6/0</td><td>Fine Science Tools</td><td>18020-60</td><td></td></tr><tr><td>Spring scissors</td><td>ROBOZ</td><td>RS-5601</td><td></td></tr><tr><td>Strabismus Scissors</td><td>Fine Science Tools</td><td>14075-09</td><td></td></tr><tr><td>Super Grip Forceps</td><td>Fine Science Tools</td><td>00649-11</td><td></td></tr><tr><td>Transparent Dressing</td><td>Cardinal Health</td><td>TD-26C</td><td></td></tr><tr><td>Triton X-1000</td><td>Fisher Scientific</td><td>AC327371000</td><td></td></tr></tbody></table>

en face preparation of mouse blood vessels

石蜡包埋组织的切片常规用于研究组织组织学和组织病理学。然而，很难从这些部分确定三维组织形态。此外，检查的组织切片可能不包含组织内为进行研究目的所必需的区域。后一种限制阻碍血管的组织病理学研究，因为血管病变以聚焦方式发展。这需要一种使我们能够从其表面到更深的区域调查血管壁的广泛区域的方法。一个完整的血管准备满足了这一要求。在本文中，我们将展示如何使小鼠主动脉和颈动脉进行面部准备，并对共聚焦显微镜和其他类型的荧光成像进行免疫荧光染色。

图3中显示了内皮的典型的面部免疫荧光图像。该图像显示了在肋间动脉（大暗蛋形区域）的开口附近获取的小鼠主动脉的单个光学部分。主动脉用抗VE-钙粘蛋白（绿色）和抗VCAM-1（血管细胞粘附分子-1）（红色）进行双重染色。每个内皮细胞在粘附连接处用绿色线性染色来概括。由于样本的微小不均匀，一些粘附点在该光学部分之外。在已知发生扰动血流的肋间动脉的开口处，抗VCAM-1染色更强。图4显示了抗VE-钙粘蛋白（绿色），抗VCAM-1（红色）和DAPI（紫色）的颈动脉的面部染色。左颈动脉（LCA）部分结扎，右颈动脉（RCA）未触动。血管标本在1天后制成<html>手术和染色。注意在结扎的血管中增加抗VCAM-1染色。用各种抗体免疫荧光染色的标本可用于研究目标蛋白的表达水平，靶蛋白的翻译后修饰的程度，当然还有内皮细胞内以及其它细胞内各种蛋白质的定位模式血管壁10,11 。 <img alt="图1" src="/files/ftp_upload/55460/55460fig1.jpg" /> 图1 :小鼠颈部区域的详细血管解剖学。在左侧显示了解剖前后的血管网。所有动脉在右图所示。黑线表示结扎。刻度:1分= 1毫米。_blank"&gt;请点击此处查看此图的较大版本。 <img alt="图2" src="/files/ftp_upload/55460/55460fig2.jpg" /> 图2:显示颈动脉和主动脉如何进入面部准备的图表。沿着血管壁的虚线表示要打开血管的切口。彩色显微照片显示实际的面部准备。刻度:1分= 1毫米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55460/55460fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55460/55460fig3.jpg" /> 图3:用抗VE-钙粘蛋白（绿色）和抗VCAM-1（红色）染色的内皮的表面图像。在肋间开口附近的内皮细胞的共焦单个光学部分是所示。注意，VCAM-1表达在位于血流非层流的血管分支点的内皮细胞中增加。使用60X（NA 1.4，油）物镜记录图像。刻度棒=20μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55460/55460fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55460/55460fig4.jpg" /> 图4:用抗VE-钙粘蛋白（绿色），抗VCAM-1（红色）和DAPI（紫色）染色的左右颈动脉的En图像。左颈动脉部分结扎，右颈总动脉完好无损。手术后24小时进行这些准备工作。与完整血管相比，连接侧VCAM-1的表达增加是明显的。这些图像是在颈总动脉分叉附近进行的使用具有60X（NA 1.4，油）物镜的激光扫描共聚焦显微镜。刻度棒=20μm。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55460/55460fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about En Face Preparation of Mouse Blood Vessels at JoVE.com

En Face Preparation of Mouse Blood Vessels

描述了制备小鼠颈动脉和主动脉的方法。当用特异性抗体进行免疫荧光染色时，这样的制剂使得我们能够研究蛋白质的定位和通过共聚焦显微镜在整个血管壁内鉴定细胞类型。

面对小鼠血液制备

Sections of paraffin embedded tissues are routinely used for studying tissue histology and histopathology. However, it is difficult to determine what the ...

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Nanyang Technological University

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Biology

En Face Preparation of Mouse Blood Vessels

20.9K 次观看.  University of Texas MD Anderson Cancer Center. 描述了制备小鼠颈动脉和主动脉的方法。当用特异性抗体进行免疫荧光染色时，这样的制剂使得我们能够研究蛋白质的定位和通过共聚焦显微镜在整个血管壁内鉴定细胞类型。 

视频: En Face Preparation of Mouse Blood Vessels

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

小鼠Spinotrapezius模型来评估的影响小动脉结扎​​术，微血管功能与重塑

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

科研

医学

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

免疫与感染

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

对小鼠脑血管净化

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

神经科学

Retinal Pericytes Visualization Using Tissue-Clearing and Fluorescent Labeling

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal vasculature. To provide a detailed view of the morphological characteristics of pericytes, this study described a new approach combining the retro-orbital injection of a fluorescent agent, immunofluorescent-staining, and tissue-clearing treatment. Firstly, the fluorescent tomato lectin was injected into the retro-orbital sinus of the live mouse to label the retinal vasculature. After 5 min, the mouse was sacrificed, and its intact retina was carefully removed from the retinal cup and immunofluorescently stained with platelet-derived growth factor receptor &#946; to reveal the pericytes. Additionally, the stained retina was treated with tissue-clearing reagent and whole mounted on the microscope slide. Through these approaches, the retinal vasculature and pericytes were clearly observed in the transparent retina. Under a confocal microscope, we obtained more opportunities to take high-resolution images for further reconstructing and analyzing the morphological characteristics of pericytes along the retinal vascular tree in a three-dimensional view. Methodologically, this protocol offers an effective approach for visualizing pericytes within the retinal vasculature, providing valuable insights into their role under both physiological and pathological conditions.

The pericytes in retinal vasculature were examined by immunofluorescent staining with platelet-derived growth factor receptor &#946; after retro-orbital injection of fluorescent tomato lectin. The labeled retina was further treated with the tissue-clearing method and whole mounted for visualizing the three-dimensional views of pericytes surrounding retinal vasculature under a confocal microscope.

Retinal pericytes are essential for vascular development and stability of the retina, playing a key role in maintaining the integrity of the retinal ...

生物化学

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

小鼠视网膜微动脉的反应性测定的制备步骤前体

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Here, a critical hindlimb ischemia experimental model is presented followed by a battery of functional, histologic and molecular tests&#160;to assess the effectiveness of angiogenic therapies.&#8203;

评估下肢缺血的Murine模型的治疗血管生成

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard ...

硬膜扩张症：血管性痴呆和阿尔茨海默病

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

作者聚焦：模拟转基因小鼠血管对阿尔茨海默病的影响

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

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 ...

面对小鼠血液制备

摘要

探索更多视频

此视频中的章节

小鼠Spinotrapezius模型来评估的影响小动脉结扎术，微血管功能与重塑

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

对小鼠脑血管净化

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

小鼠视网膜微动脉的反应性测定的制备步骤前体

评估下肢缺血的Murine模型的治疗血管生成

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

使用 En 面免疫荧光染色直接观察血管内皮细胞

面对小鼠血液制备

摘要

探索更多视频

此视频中的章节

小鼠Spinotrapezius模型来评估的影响小动脉结扎​​术，微血管功能与重塑

隔离容器的血管扩张和细胞外基质的紧皮肤小鼠隔离

对小鼠脑血管净化

3D Visualization of Retinal Vascular Pericytes in Mice by Immunostaining

小鼠视网膜微动脉的反应性测定的制备步骤前体

评估下肢缺血的Murine模型的治疗血管生成

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

一种诱导小鼠脑裂症以模拟大血管介导的脑血管功能障碍的视觉方法

使用 En 面免疫荧光染色直接观察血管内皮细胞

小鼠Spinotrapezius模型来评估的影响小动脉结扎术，微血管功能与重塑