这项工作是由威康信托基金和英国心脏基金会的资助。

所有步骤都在室温下进行，除非另有说明。 1。产后的眼睛剜除术和固定<ol><li>将人道扑杀鼠标小狗在其一侧，清除皮肤覆盖在眼睛上，用剪刀。 </li><li>使用剪刀和镊子，enucleate眼睛。 </li><li>将剪刀低于去核的眼睛，切开视神经及周围组织，并取出眼球。转移至24孔板中，含有4％多聚甲醛（PFA），在室温下在2×PBS。 </li><li>允许每只眼睛定为10 - 15分钟，然后转移到寒冷的2倍PBS在冰5 - 10分钟。这是最好错开切除，和随后的固定，以确保固定的程度等于为每只眼睛。 </li></ol> 2。产后小鼠视网膜解剖<ol><li>传输到培养皿中含有2倍PBS眼中已削减到扩大缸径和下DISSE的地方用塑料巴斯德吸管cting显微镜。 </li><li>眼部周围使用镊子和剪刀移除任何脂肪。 </li><li>皮尔斯在角膜边缘锋利的剪刀，切断周围的角膜和虹膜和丢弃。 </li><li>插入视网膜和巩膜之间的镊子，小心不要撕裂视网膜删除巩膜。脉络膜层也可以被除去，但是，如果有一个在此步骤中发生的视网膜损害的风险，可以留在脉络膜层，因为它不应该显着影响的免疫染色的质量。 </li><li>取下镜头并使用镊子玻璃体。 </li><li>删除的hyaloids血管中心的眼睛聚集在一起，用细镊子，然后迅速拉离视网膜中心（或者玻璃体血管细剪刀剪断基地）。 </li><li>冲洗视网膜PBS在一个干净的培养皿杯形。 </li><li>进行4〜5个放射状切口，达到约2/3半径的视网膜使用弹簧剪刀RS创建一个“花瓣”的形状。 </li><li>绘制PBS使用巴斯德吸管展平视网膜，然后用一小块吸水纸去除任何多余的PBS。 </li><li>吸管慢慢地冷（-20℃）甲醇一滴一滴到视网膜表面，直至完全覆盖，然后提供额外的甲醇洪水。这将有助于修复视网膜，并将促进通透性。视网膜会变成白色。 </li><li>转移到2毫升，已被切割拓宽孔用塑料巴斯德吸管管。 </li><li>在寒冷的甲醇离开视网膜至少20分钟，然后再继续进行染色。这里所用的抗体中，只有VE-钙粘蛋白不能被成功地使用甲醇固定的视网膜。在这种情况下，视网膜需要进行免疫染色步骤9之后的直路。 </li><li>如果需要的话，视网膜可以被存储在甲醇中在-20℃下染色前的数月。 </li></ol> 3。免疫/凝集素染色的视网膜仍有合适的lature <ol><li>小心取出/倒出甲醇和轻轻冲洗视网膜短暂PBS（在这个阶段，视网膜仍处于相同的2毫升管从上面的步骤11）。 </li><li>删除PBS和盖视网膜100微升彼尔姆/块解决方案（PBS + 0.3％的Triton + 0.2％BSA）+适当的血清5％（ 见表1），轻轻摇晃1小时。 </li><li>删除权限/座和孵化视网膜轻轻摇动过夜，4℃，100微升（ 见表1）选定的主抗体和/或IsolectinB4，所有准备在彼尔姆/座稀释。 </li><li>取出抗体/凝集素和洗视网膜4×10分钟，PBS + 0.3％TRITON（PBSTX）的。 </li><li>在某些情况下， 例如，下面的染色IsolectinB4的ALEXA488或一个直接结合的一次抗体，第二抗体是不必需的，可以直接安装在视网膜（步骤7）。当未结合的初级抗体被用来在步骤3中，然后加入100μl适当的氟管三基色的第二抗体（稀释1/200 PBSTX）加入时间在4℃下，或在室温下搅拌4小时孵育过夜。 </li><li>拆下二次抗体和视网膜4×15分钟洗在2毫升PBSTX中。 </li><li>视网膜上的幻灯片，使用大口径塑料巴斯德吸管转移和删除多余的PBSTX与吸收剂组织。通过加入50μl的装载视网膜延长封固到盖玻片，轻轻地在视网膜上的位置。 </li><li>转移滑动，在4℃冰箱过夜，以确保它们是平坦的，避光，允许延长封固设置。 </li><li>图片视网膜使用共聚焦显微镜或落射荧光显微镜。 </li></ol>

<ol>
	<li>Carmeliet, P., Jain, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+and+clinical+applications+of+angiogenesis.">Molecular mechanisms and clinical applications of angiogenesis.</a> Nature. 473 (7347), 298-307 (2011).</li><li>Staton, C. A., 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=Current+methods+for+assaying+angiogenesis+in+vitro+and+in+vivo.">Current methods for assaying angiogenesis in vitro and in vivo.</a> Int J. Exp. Pathol. 85 (5), 233-248 (2004).</li><li>Goodwin, A. 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Biol. 248 (2), 307-318 (2002).</li><li>Fruttiger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+retinal+vasculature.">Development of the retinal vasculature.</a> Angiogenesis. 10 (2), 77-88 (2007).</li><li>Sawamiphak, S., Ritter, M., Acker-Palmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+retinal+explant+cultures+to+study+ex+vivo+tip+endothelial+cell+responses.">Preparation of retinal explant cultures to study ex vivo tip endothelial cell responses.</a> Nat. Protoc. 5 (10), 1659-1665 (1038).</li><li>Mahajan, V. B., Skeie, J. M., Assefnia, A. H., Mahajan, M., Tsang, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+eye+enucleation+for+remote+high-throughput+phenotyping.">Mouse eye enucleation for remote high-throughput phenotyping.</a> J. Vis. Exp. (57), e3184 (2011).</li><li>Pitulescu, M. E., Schmidt, I., Benedito, R., Adams, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+gene+targeting+in+the+neonatal+vasculature+and+analysis+of+retinal+angiogenesis+in+mice.">Inducible gene targeting in the neonatal vasculature and analysis of retinal angiogenesis in mice.</a> Nat. Protoc. 5 (9), 1518-1534 (2010).</li><li>Allinson, K. R., Lee, H. S., Fruttiger, M., McCarty, J., Arthur, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+expression+of+TGFbeta+type+II+receptor+is+required+to+maintain+vascular+integrity+during+postnatal+development+of+the+central+nervous+system.">Endothelial expression of TGFbeta type II receptor is required to maintain vascular integrity during postnatal development of the central nervous system.</a> PLoS One. 7 (9), e39336 (2012).</li><li>Mahmoud, M., 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=Pathogenesis+of+arteriovenous+malformations+in+the+absence+of+endoglin.">Pathogenesis of arteriovenous malformations in the absence of endoglin.</a> Circ. Res. 106 (8), 1425-1433 (2010).</li><li>Lebrin, F., 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=Thalidomide+stimulates+vessel+maturation+and+reduces+epistaxis+in+individuals+with+hereditary+hemorrhagic+telangiectasia.">Thalidomide stimulates vessel maturation and reduces epistaxis in individuals with hereditary hemorrhagic telangiectasia.</a> Nat. Med. 16 (4), 420-428 (2010).</li><li>Pi, L., 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=Role+of+connective+tissue+growth+factor+in+the+retinal+vasculature+during+development+and+ischemia.">Role of connective tissue growth factor in the retinal vasculature during development and ischemia.</a> Invest. Ophthalmol. Vis. Sci. 52 (12), 8701-8710 (2011).</li><li>Powner, M. B., 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=Visualization+of+gene+expression+in+whole+mouse+retina+by+in+situ+hybridization.">Visualization of gene expression in whole mouse retina by in situ hybridization.</a> Nat. Protoc. 7 (6), 1086-1096 (2012).</li></ol>

作者宣称，他们有没有竞争经济利益。

这里介绍的方法提供了一个简单而有效的方式来获取图像新生小鼠视网膜染色整装准备，使得它容易量化和生理相关的血管生成模型。最初，老鼠的眼睛是相当困难的，由于其体积小，球形，解剖，所以一些做法和良好的清扫工具都需要可靠的结果。夹层2倍浓度的PBS中制备的眼睛是很重要的，因为这会导致少量的水从眼睛损失，并降低的轨道内的压力，这有利于夹层。良好的准备是非常重要的视...

血管生成是一种复杂的发育过程中，定义通过从预先存在的血管网形成新的血管，并通过多条信号通路的调节。其中最突出的是VEGF通路。血管内皮生长因子释放由缺血性细胞，导致血管生成的启动，在相邻的血管发芽。领先的内皮细胞从一个新的血管芽是&#39;冰山&#39;细胞，产生对VEGF的来源，伸出伪足，其次是&#39;茎&#39;内皮细胞增殖。在这种方式中，活化的内皮细胞迁移和增殖实现的无血管区域，在那里形成新血管管。为了稳定，以支持这些管子的血流量，血管内皮细胞与周皮细胞和平滑肌细胞，围绕新的血管1。胚胎的血管形成及组织生长的血管生成是必不可少的。然而，它也有助于许多pathologi卡尔疾病，如癌症，而在血管生成中的缺陷与缺血性疾病。因此，开发有效的药物治疗作为治疗疾病的一种手段，是调节血管生成的极大兴趣。例如，有效的抗血管生成因子将减少癌的生长，而促血管生成的策略可以用于治疗缺血性疾病。因此，必须更好地了解新血管的形成和开发新的治疗策略，以提高或降低血管生长调节血管生成模型。 血管新生研究的基本要素是选择一个合适的血管模型。许多体外模型的目的是为了再现血管生成的基本步骤，如血管内皮细胞的迁移，增殖和存活2,3。 在体外试验中经常使用的一种是有支链的网络上的基底膜基质的血管内皮细胞的形成，虽然吨他的是不特定的内皮细胞，也不会通常包含周细胞或平滑肌细胞的支持。评估体外芽生式血管生成，如胚体的测定，主动脉环测定和胎 ​​儿跖骨检测小鼠器官培养允许的上下文中的额外的支持细胞，血管内皮细胞的血管生成的分析。然而，这些检测方法的主要限制包括血流量不足，并随着时间的推移回归的船只，给一个有限的窗口进行分析。因此，更准确地了解血管生成的调控， 在体内模型中是必需的，如那些在小鼠皮下植入的海绵或基底膜插头开发，以及后肢缺血模型。然而，这些模型是具有挑战性的分析，由于复杂的三维结构的新生血管。与此相反，已证明斑马鱼胚胎的一个很好的模型可视化的血管生成在整个生物体4。然而，进化更密切相关的人类比斑马鱼鼠标，让鼠标在许多情况下，一款首选机型。因此，出生后小鼠视网膜血管生成是一个很好的特点的血管生成研究的首选方法。它不仅提供了生理的设置，但也是一个二维的血管丛，可以很容易地可视化使用适当的荧光染料。血管网，血管内皮细胞增殖，发芽，血管周围细胞的招募和血管重塑的建筑都可以使用这种技术研究。 在新生小鼠视网膜，视网膜血管的发展发生在出生后的第一周。小鼠与人类不同的是，是天生的盲人和视网膜血管的出生后。视网膜血管产生的视网膜的中心靠近视神经在出生后的第0天（P0），血管生成和发展，形成高度组织ð血管丛到达视网膜周边约P8 5。在其特有的方式与毛细管丛逐渐成长从视盘向外朝向的外周，以产生一个有规律的交替模式具有介于其间的毛细血管网的动脉和静脉血管的发展。视网膜血管提供了一个理想模型来研究血管新生的血管可以很容易地确定，因为他们成长在什么本质上是一个二维平面，从而使得它比较容易检查视网膜血管台安装准备5。已报道的方法，在数小时内体外内皮小费细胞反应进行分析隔离小鼠新生儿的视网膜6。另外，需要更详细的调查整个视网膜血管和相关的血管标记免疫组的视网膜固定的标本在不同的发展阶段。 在这里，我们提供详细描述了一个可靠的和技术上直线前进的方法，我们使用整装染色小鼠视网膜血管丛，从最初的眼球摘除产后眼（7）通过染色协议到最终成像显微镜下。

<table border="1">
	<tbody>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Material</td>
		</tr>
		<tr>
			<td>Plastic pipettes 3 ml</td>
			<td>Scientific Laboratory Supplies</td>
			<td>PIP4210</td>
			<td>Remove tip with scissors to create a wide bore</td>
		</tr>
		<tr>
			<td>BD Falcon 24-well plates</td>
			<td>Scientific Laboratory Supplies</td>
			<td>353226</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Select Petri dishes TV 90 mm</td>
			<td>Scientific Laboratory Supplies</td>
			<td>SLS2002</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Histobond slides</td>
			<td>VWR</td>
			<td>631-0624</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Coverslips 22 x 22 mm</td>
			<td>VWR</td>
			<td>631-1336</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dumont Tweezers #5</td>
			<td>World precision instruments</td>
			<td>14095</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spring scissors 10.5 cm long, with straight 8 mm blades</td>
			<td>World precision instruments</td>
			<td>501235</td>
			<td>Used for enucleation</td>
		</tr>
		<tr>
			<td>Vannas scissors 8.5 cm long, with straight 7 mm blades, and 0.025 x 0.015 mm superfine tips</td>
			<td>World precision instruments</td>
			<td>500086</td>
			<td>Used for dissecting retina</td>
		</tr>
		<tr>
			<td>Superfine vannas scissors 8 cm long with straight 3 mm blades, and 0.015 x 0.015 mm superfine tips</td>
			<td>World precision instruments</td>
			<td>501778</td>
			<td>Used for dissecting retina</td>
		</tr>
		<tr>
			<td>crystal clear' tubes 2 ml</td>
			<td>Starlab</td>
			<td>E1420-2000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Reagents</td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma</td>
			<td>P5655</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td>PBS reagent</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Make up in 2x PBS and store at -20 &deg;C</td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7638</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T9284</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>Sigma</td>
			<td>D9663</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Prolong Gold anti-fade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Mounting reagent</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-alexa 594</td>
			<td>Invitrogen</td>
			<td>I21413</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4-alexa 488</td>
			<td>Invitrogen</td>
			<td>I21411</td>
			<td>Use at 1:200 dilution</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Primary Antibodies</td>
		</tr>
		<tr>
			<td>Anti-NG2 (rabbit polyclonal )</td>
			<td>Millipore</td>
			<td>AB5320</td>
			<td>Detects pericytes</td>
		</tr>
		<tr>
			<td>Anti-GFAP (clone GA5, mouse monoclonal)</td>
			<td>Millipore</td>
			<td>MAB360</td>
			<td>Detects astrocytes</td>
		</tr>
		<tr>
			<td>Anti-Desmin (clone Y66, rabbit monoclonal)</td>
			<td>Millipore</td>
			<td>04-585</td>
			<td>Detects pericytes</td>
		</tr>
		<tr>
			<td>Anti-Collagen IV (rabbit polyclonal )</td>
			<td>Chemicon</td>
			<td>AB756P</td>
			<td>Detects vascular basement membrane</td>
		</tr>
		<tr>
			<td>Anti-alpha smooth muscle actin-Cy3 (clone 1A4, mouse monoclonal)</td>
			<td>Sigma</td>
			<td>C6198</td>
			<td>Detects vascular smooth muscle cells.</td>
		</tr>
		<tr>
			<td>Anti-Endoglin (clone MJ7/18, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>550546</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>Anti-CD31 (clone MEC13.3, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>553370</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>Anti-VE-Cadherin (clone 11D4.1, rat monoclonal)</td>
			<td>BD Pharmingen</td>
			<td>550548</td>
			<td>Detects endothelial cell junctions</td>
		</tr>
		<tr>
			<td>Anti-Alk1 (goat polyclonal)</td>
			<td>R&amp;D Systems</td>
			<td>AF770</td>
			<td>Detects endothelial cells</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Secondary Antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-rabbit-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11012</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-rat-Alexa488</td>
			<td>Invitrogen</td>
			<td>A11006</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-rat-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11007</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Goat anti-mouse-Alexa488</td>
			<td>Invitrogen</td>
			<td>A11029</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>Donkey anti-goat-Alexa594</td>
			<td>Invitrogen</td>
			<td>A11058</td>
			<td>All secondary antibodies are aliquoted immediately on arrival and stored at -20 &deg;C.</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Microscopes</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Zeiss</td>
			<td>Stemi SV6</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Zeiss</td>
			<td>Axiocam HRc</td>
			<td>Camera for dissection microscope</td>
		</tr>
		<tr>
			<td>Epifluorescent Microscope</td>
			<td>Zeiss</td>
			<td>Axioimager with Apotome</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Zeiss</td>
			<td>Axiocam HRm</td>
			<td>Camera for Axioimager</td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1R</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

whole mount immunofluorescent staining of the neonatal mouse retina to investigate angiogenesis in vivo

血管生成是定义新的血管形成新血管从预先存在的血管网发芽的复杂过程。血管生成中起着关键作用不仅在器官和组织的正常发育，而且在许多疾病中，血管的形成是失调，如癌症，失明和局部缺血性疾病。在成年生活中，血管通常是静态的，因此血管生成是尝试和调节新血管的形成，特别是在疾病新型药物开发的重要目标。为了更好地了解血管新生，并制定相应的战略来调节，模型，准确地反映了不同的生物所涉及的步骤。小鼠视网膜新生血管生成提供了一个很好的模型，因为动脉，静脉和毛细血管的发展在出生后的第一周，形成血管丛。该模型还具有的优点是具有双 - 二mensional（2D）结构简单的决策分析较复杂的三维解剖其他血管网络。通过分析视网膜血管丛出生后在不同的时间，它是在显微镜下可以观察到的血管生成的各个阶段。本文演示了一个简单的流程分析荧光染色法，凝集素和血管特异性抗体的小鼠视网膜血管。

解剖后的视网膜应该看起来像平坦的花（ 图1）。通过使用上文所述的协议，使用凝集素B4 ALEXA488染色和支持血管平滑肌细胞，用抗α-平滑肌肌动蛋白（ 图2），可以看出，内皮细胞。低功耗的图，使用图2中的5倍的目标允许的视网膜血管组织的概述。此外，通过使用不同的以上的协议在给定的抗体相结合，内皮细胞，用抗CD31免疫染色使用抗NG2（ 图3），...

Watch this Scientific Journal Video about Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo at JoVE.com

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

新生小鼠视网膜提供了一个很好的特点生理血管生成模型，它允许不同的基因或药物，在调节血管生成的角色的调查<em&gt;在体内</em&gt;上下文。免疫荧光染色准确地显示血管丛是这些类型的研究成功的关键。

整装免疫荧光染色新生小鼠视网膜血管生成调查<em&gt;在体内</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

whole-mount-immunofluorescent-staining-neonatal-mouse-retina-to

Research

JoVE Journal

Neuroscience

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

45.3K 次观看.  Newcastle University. 新生小鼠视网膜提供了一个很好的特点生理血管生成模型，它允许不同的基因或药物，在调节血管生成的角色的调查在体内上下文。免疫荧光染色准确地显示血管丛是这些类型的研究成功的关键。

视频: Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

在全安装的胚胎神经系统的可视化果蝇胚胎

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

科研

神经科学

Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.

A formal demonstration of the dissection of a mouse eye, resulting in a whole mount of the retinal pigment epithelium.

解剖鼠标眼的视网膜色素上皮的全山

The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

在发展中国家小鼠视网膜体内电穿孔

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

小鼠胚胎感觉轴突预测整个安装成像

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

全山解剖和成年小鼠耳蜗的免疫

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

Patch Clamp Recording of Starburst Amacrine Cells in a Flat-mount Preparation of Deafferentated Mouse Retina

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic network, electrophysiological recordings are frequently used to study connections among individual neurons. We have optimized a flat-mount preparation for patch clamp recording of genetically marked neurons in both GCL (ganglion cell layer) and INL (inner nuclear layer) of mouse retinas. Recording INL neurons in flat-mounts is favored over slices because both vertical and lateral connections are preserved in the former configuration, allowing retinal circuits with large lateral components to be studied. We have used this procedure to compare responses of mirror-partnered neurons in retinas such as the cholinergic starburst amacrine cells (SACs).

This protocol demonstrates how to perform whole-cell patch clamp recording on retinal neurons from a flat-mount preparation.

星爆无长突细胞的膜片钳在Deafferentated小鼠视网膜的平板安装准备

The mammalian retina is a layered tissue composed of multiple neuronal types. To understand how visual signals are processed within its intricate synaptic ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

一个简单的一步解剖协议成人全安装准备<em&gt;果蝇</em&gt;脑

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Whole-Mount Staining, Visualization, and Analysis of Fungiform, Circumvallate, and Palate Taste Buds

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells communicate with nerve fibers that carry this information to the brain. Because taste-transducing cells continuously die and are replaced throughout adulthood, the taste-bud environment is both complex and dynamic, requiring detailed analyses of its cell types, their locations, and any physical relationships between them. Detailed analyses have been limited by tongue-tissue heterogeneity and density that have significantly reduced antibody permeability. These obstacles require sectioning protocols that result in splitting taste buds across sections so that measurements are only approximated, and cell relationships are lost. To overcome these challenges, the methods described herein involve collecting, imaging, and analyzing whole taste buds and individual terminal arbors from three taste regions: fungiform papillae, circumvallate papillae, and the palate. Collecting whole taste buds reduces bias and technical variability and can be used to report absolute numbers for features including taste-bud volume, total taste-bud innervation, transducing-cell counts, and the morphology of individual terminal arbors. To demonstrate the advantages of this method, this paper provides comparisons of taste bud and innervation volumes between fungiform and circumvallate taste buds using a general taste-bud marker and a label for all taste fibers. A workflow for the use of sparse-cell genetic labeling of taste neurons (with labeled subsets of taste-transducing cells) is also provided. This workflow analyzes the structures of individual taste-nerve arbors, cell type numbers, and the physical relationships between cells using image analysis software. Together, these workflows provide a novel approach for tissue preparation and analysis of both whole taste buds and the complete morphology of their innervating arbors.

This paper describes methods for tissue preparation, staining, and analysis of whole fungiform, circumvallate, and palate taste buds that consistently yield whole and intact taste buds (including the nerve fibers that innervate them) and maintain the relationships between structures within taste buds and the surrounding papilla.

真菌形态、环形和味觉味蕾的全镶嵌染色、可视化和分析

Taste buds are collections of taste-transducing cells specialized to detect subsets of chemical stimuli in the oral cavity. These transducing cells ...