作者想感谢最初帮助建立的器官文化和技术援助的美国Laub和美国Gerster E.德拉罗萨和人工智能Valenciano。

所有的设备和试剂必须购买无菌或需要加热或蒸汽消毒或用70％乙醇消毒。 作者状态，对动物的实验是根据欧洲共同体理事会指令（86/609/EEC），美国国立卫生研究院的机构动物所规定的实验程序和法规的动物护理和使用准则保养和使用委员会（IACUC）在杜伊斯堡 - 埃森大学（德国）。 第1部分：不同发育阶段的小鼠的眼睛剜除剜除的胚胎眼睛<ol><li>一次怀孕交配设立和指定的女性交配检测阴道塞的一天上午妊娠0天。 </li><li>女性怀孕时，胚胎的发展已达到了预期的阶段（这里是：胚胎一天（五）15）和蜡板6固定颈椎脱位处死。 </li><li>腹壁蘸70％乙醇，沿中线切开皮瓣是固定的横向引脚6。 </li><li> uterusses是从腹部，用冷PBS 6分离并转移到烧杯中删除。 </li><li>胚胎分离，转移到培养皿中，子宫壁和胚胎膜由使用镊子6小心删除。 </li><li>胚胎死于斩首。 </li><li>摘除眼睛是被罚款，弯钳的使用，“剥皮”的眼睛从眼窝。 </li></ol>剜除术，产后和成人的眼睛<ol start="1" type="1"><li>年轻的幼崽被杀害断头，年龄较大的幼崽和成人颈椎脱位。 </li><li> P15产后阶段，要机械地使用镊子打开和扩大两个带弹簧剪刀眼睑横向削减的时间点时，小鼠睁开眼睛，眼睛开衩。 </li><li>眼睛被摘除弯钳的帮助下，施加压力的轨道。 </li></ol>注：在出生后第2天，眶骨软骨，重要的是不适用的压力太大，而试图消除眼睛。 相比之下，在成年小鼠，眶骨是坚定的。因此，为了enucleate的眼睛，它是足够的申请，恕不提前扩大眼开衩对轨道的压力。 第2部分：胚胎，产后和成年鼠的视网膜的解剖剖析视网膜<ol><li>眼睛是放置在一个小的Petri菜用无菌PBS和周围的眼睛层下解剖镜下删除。 </li><li>为了消除外眼层，在产​​后阶段，成人的眼睛，视神经已被切断钳由春天的剪刀或挤压关闭的帮助下，尽量接近尽可能的基础。 </li><li>转动眼球，使视神经原先居住的孔的背面朝向您。输入之间的视网膜和视网膜色素上皮细胞与两个非常精细的视神经侵入眼层的地点钳的技巧空间。 注：一般情况下，色素上皮细胞可以很容易地确定深色。这种色素层可能在一些小鼠突变株 - 尤其是在白化动物 - 然而，没有着色，因而可能不容易被检测到。 </li><li>通过仔细地向两侧撕裂两个钳，删除附加脉络膜膜和巩膜色素上皮细胞。 </li><li>剥离层角膜水平，然后打开眼罩，镜头一侧，消除色素上皮，脉络膜膜和巩膜角膜，而其余的视网膜杯其他镊子。 </li><li>小镜头掌握玻璃体和钳撕裂的同时，保持第二镊子视网膜wholemount。 注：解剖胚胎的眼睛时，请务必彻底清除玻璃体下方的三角形，帐篷状毛细血管丛玻璃体。 在成人眼，玻璃体需要双方在把握和护理钳提示应采取不捅破玻璃体，其内容是粘性和支镊子，阻碍了其去除。 </li><li>器官wholemount文化的视网膜的杯子收集在一个96孔的板含有200μL贝科改良Eagle培养基（见下文）。 </li></ol>注：个人的视网膜之间的清扫，保持96孔收集板含有培养基中，培养基的pH值在孵化器是由碳酸盐系统通过触发的CO 2。 第3部分：小鼠器官视网膜wholemount文化<ol><li>准备中/加权7.8克贝科的修改鹰的营养混合物的F - 12火腿（DMEM）和碳酸氢钠30.6克MiIliQ水都溶解在500毫升的文化。调整pH值至7.15。引擎盖下添加50毫克的apo - transferin，50μLputrescin（股票：60mg/ml），和2.5毫升的庆大霉素（200毫米），50μL亚硒酸钠（股票：：52μg/ml），孕酮（60μg/ml股票）。混合通过一个瓶子的顶部过滤器和过滤。使用前立即加入每毫升培养液中加入10μl谷氨酰胺（200毫米）。 注：血清胰岛素无介质可以被保存在4 ° C至2周，并诱导细胞凋亡的实验中使用的，因为没有胰岛素抵消的影响。如果需要超过24小时潜伏期较长的wholemounts将不会评估和细胞死亡率，血清胰岛素（如小牛血清; FCS）或补充应补充，以提高生存率。 </li><li>开始之前的文化，视网膜wholemounts预孵育15分钟在37 ° C与200μL温，含有0.5mg/ml的透明质酸酶预消化包含内部和外部的限制并购LLER神经胶质细胞的细胞膜的透明质酸酶的酸碱平衡的DMEM细胞，促进外源物质的渗透。 </li><li>视网膜被转移到24孔板2毫升贝科化学定义的改良Eagle培养基几个尽可能和作为器官wholemounts培养的透明质酸。 注：对于转让的视网膜，使用1毫升吸管和切割枪头几毫米，扩大开放。对于胚胎的眼睛，200μL枪头就足够了。切割边缘应理顺第二枪头插入和扭曲。 24-48小时的短期文化，所有的步骤都可以在替补席上，但如果对文化的污染是一个问题，一个引擎盖下的工作。 </li><li>文化是保持在37 ° C 5％的CO 2气氛中为24 - 48小时，如药物治疗与重组因素。 </li></ol>第4部分：解离培养的视网膜wholemounts <ol><li>所需的培养时间后，视网膜被收集在圆底2毫升的Eppendorf管含850μLPBS和50μL牛血清白蛋白（BSA; 30毫克/毫升）。 </li><li> Eppendorf管与视网膜放置到一个加热块加热至37 ° C。 </li><li>每个Eppendorf管中加入25μl的胶原酶（200 U /毫升）和25μL透明质酸酶（20mg/ml），并开始解离成单细胞悬液3张通行证通过硅化巴斯德吸管视网膜。 </li><li>加入10μL胰蛋白酶（1mg/ml的），等待3-5分钟，然后慢慢地吸液管的3-5倍，并与硅化巴斯德吸管机械地分解组织。 </li><li>添加10μLDNA酶I（5mg/ml），再等待3-5分钟，然后慢慢的吸液管的3-5倍和硅化巴斯德吸管。 注：酶解培养时间各不相同，取决​​于眼睛和发育阶段的大小，分别。检查通过向上和向下轻轻吹打组织酶消化阶段。 </li><li>如果细胞悬液现在不是同质，但仍然包含了主要的细胞聚集，添加额外的10μL胰蛋白酶和10μLDNA酶一</li><li>当细胞悬液是同质的，停止消化的组织是由另外10μLEDTA（0.5米），Eppendorf管加热器和细胞悬浮液中删除除了1毫升新鲜，冰冷的8％多聚甲醛固定为1h （PFA）在室温旋转振动筛。 </li></ol>第5部分：游离细胞悬液洗涤<ol><li>细胞悬液，离心5分钟，4 ° C和0.2 RCF，在冷却离心机。 </li><li>被丢弃上清液，沉淀在1毫升PBS含有3mg/ml BSA重悬。 </li><li>沉淀两次重复这些洗涤步骤后，终于重新悬浮于500μLPBS中3mg/ml牛血清白蛋白，5MM EDTA和0.1％的叠氮化钠。 </li></ol>注：钠酸除了让数天在4 ° C储存的细胞悬液但是，如果免疫细胞化学染色将遵循， 不添加钠酸的再悬浮缓冲，染色质量损失结果。 第6部分：定量细胞凋亡和增殖分析细胞悬液Cytospin <ol><li>磨砂高端显微镜玻片上，一个cytospin过滤器与一个或两个孔和cytospin漏斗插入cytospin幻灯片剪辑。幻灯片剪辑关闭，并在cytospin转子的位置。 </li><li>匀浆向上和向下轻轻吹打分离的细胞悬液。 注：根据视网膜的发育阶段，从解离过程中的细胞悬液可能需要用PBS稀释，获得的细胞计数。 </li><li>一个细胞悬液等份（100μL）是适用于一个cytospin漏斗。 注意：当吹打细胞悬浮液漏斗，吸管的尖端应达到一路下降漏斗底部。重要的是通过吸液管的第二个压力点不推，因为这将创建气泡的cytospin后，这将是在细胞现货中可见，阻碍细胞计数。 </li><li>细胞悬液，发现在7分钟700转的幻灯片。 </li><li>细胞凋亡水平的外源因素的影响的决心，可染色与4&#39;,6 - Diamidino - 2 -苯基吲哚（DAPI;2μg/ml），用荧光安装介质安装。在细胞凋亡的变化，可通过计数cytospin细胞点至少1000个细胞（包括至少10 pycnotic核）和细胞死亡率细胞总数的百分比计算计数3,4。 注 ：此外，凋亡细胞核的分布，可以在体外培养的视网膜 wholemounts flatmounts 3或低温恒温器sections4评估TdT介导的dUTP缺口末端标记法（ TUNEL）。 </li><li>检测细胞的增殖，BrDU（5微米）可以被添加6H的文化和BrdU掺入年底前，在可视化细胞匀浆cytospins由免疫细胞化学染色使用抗BrdU抗体（如发育研究杂交瘤细胞银行，爱荷华州，美国）。 </li><li>在不同的视网膜细胞类型的治疗效果，可在cytospins可视Brn3a（神经节细胞标记物）或视蛋白（感光器标记）和用DAPI counterstaining神经元特异性抗体。 </li></ol>第7部分：代表结果<img src="/files/ftp_upload/1634/1634fig1tmb.jpg" border="0" alt="图1" /> 图1：在小鼠器官视网膜wholemounts的准备步骤 双眼头的鼠标。B与镜头一侧的小鼠眼，仍然存在的所有层。C从鼠眼视神经背面还连着 。与鼠眼巩膜和色素上皮部分切除，E小鼠视网膜角膜，巩膜和色素上皮细胞完全清除，但仍然存在的镜头和玻璃体。F小鼠晶状体和玻璃体视网膜wholemount杯删除。请<a href="https://www.jove.com/files/ftp_upload/1634/1634fig1.jpg">点击这里</a>看到图1的放大版本。 <img src="/files/ftp_upload/1634/1634fig2tmb.jpg" border="0" alt="图2" /> 图2：cytospin和部分器官视网膜的wholemount文化分析 分析细胞凋亡，细胞悬浮液的分离cytospins经DAPI染色和pycnotic原子核可以通过核碎裂或染色质凝聚的区别开来（在箭头）。另外，wholemount节（CE;小鼠视网膜日龄（P））或视网膜flatmount（F）的可承受一个TUNEL法检测，并用DAPI（五）counterstained 。在不同的视网膜细胞类型的治疗效果，可以可视化在cytospins 协鑫像标记Brn3a（在B 的箭头 ），神经节细胞神经元特异性抗体，神经节细胞层;。INL的，前瞻性的内核层。请<a href="https://www.jove.com/files/ftp_upload/1634/1634fig2.jpg">点击这里</a>看大图图2。

<ol>
	<li>Rieke, M., Gottwald, E., Weibezahn, K. -. F., Layer, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+reconstruction+in+3D-spheroids+from+rodent+retina+in+a+motion-free,+bioreactor-based+microstructure.">Tissue reconstruction in 3D-spheroids from rodent retina in a motion-free, bioreactor-based microstructure.</a> Lab. Chip. 8, 2006-2213 (2008).</li><li>Donovan, S. L., Dyer, 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=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nature Protocols. 1, 2710-2718 (2006).</li><li>Duenker, N., Valenciano, A. I., Franke, A., Hernandez-Sanchez, C., Dressel, R., Behrendt, M., de Pablo, F., Krieglstein, K., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balance+of+pro-apoptotic+transforming+growth+factor-beta+and+anti-apoptotic+insulin+effects+in+the+control+of+cell+death+in+the+postnatal+mouse+retina.">Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.</a> Eur. J. Neurosci. 22, 28-38 (2005).</li><li>Franke, A. G., Gubbe, C., Beier, M., Duenker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factors+beta+and+Bone+morphogenetic+proteins:+Cooperative+players+in+chick+and+murine+programmed+retinal+cell+death.">Transforming growth factors beta and Bone morphogenetic proteins: Cooperative players in chick and murine programmed retinal cell death.</a> J. Comp. Neurol. 495, 263-278 (2005).</li><li>de la Rosa, E. J., D&#237;az, B., De Pablo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoculture+of+the+chick+embryonic+neuroretina.">Organoculture of the chick embryonic neuroretina.</a> Curr. Top. Dev. Biol. 36, 133-144 (1998).</li><li>Dohle, D. S., Pasa, S. D., Gustmann, S., Laub, M., Wissler, J. H., Jennissen, H. P., Duenker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+How+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: How it really works.</a> J Vis Exp. 32, (2009).</li></ol>

利用小鼠器官视网膜wholemount文化2-5以上的解离，单层，retinospheroid或重新聚合的3D球体在保存神经元的相互作用和连接谎言文化，模仿体内的情况。 2前报告相比，我们的视频的文章提供了一个在剜除小鼠的眼睛，包括晶状体和玻璃体切除不损害视网膜的不同发育阶段的视网膜剥离的特殊性详细演示。晶状体和玻璃体切除药理操作至关重要，既阻碍物质的视网膜层的访问。?...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>Tool</td>
<td>ZEISS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile</td>
</tr>
<tr>
<td>Dulbecco`s modified eagle`s medium / nutrient mixture F-12 Ham</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 8900</td>
<td>DMEM / F-12</td>
</tr>
<tr>
<td>Apo-transferin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T 1147</td>
<td>&nbsp;</td>
<tr>
<td>Putrescin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P 5780</td>
<td>&nbsp;</td>
<tr>
<td>Sodium selenite</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S 9133</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P 6149</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-024</td>
<td>200 mM (100X), liquid</td>
</tr>
<tr>
<td>Bovine serum albumine (BSA)</td>
<td>Reagent</td>
<td>Roth</td>
<td>8076.3</td>
<td>30 mg/ml</td>
</tr>
<tr>
<td>Collagenase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C 0773</td>
<td>200 U/ml</td>
</tr>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T4799</td>
<td>From porcine pancreas; 1 mg/ml</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H 3884</td>
<td>200 mg/ml</td>
</tr>
<tr>
<td>DNase I</td>
<td>Reagent</td>
<td>Roche</td>
<td>1 284 932</td>
<td>10 mg/ml</td>
</tr>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E 6511</td>
<td>&nbsp;</td>
<tr>
<td>Silicone solution</td>
<td>Reagent</td>
<td>Serva</td>
<td>35130</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>8% PFA in 0.1M phosphate buffer (pH 7.4).</td>
</tr>
<tr>
<td>4',6-diamidino-2-phenylindole dihydrochloride</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 0542</td>
<td>DAPI</td>
</tr>
<tr>
<td>Fluorescent Mounting Medium</td>
<td>Reagent</td>
<td>Dako</td>
<td>S3023</td>
<td>&nbsp;</td>
<tr>
<td>BrDU</td>
<td>Reagent</td>
<td>Sigma</td>
<td>B 9285</td>
<td>&nbsp;</td>
<tr>
<td>96-well plates</td>
<td>Tool</td>
<td>FALCON</td>
<td>3072</td>
<td>&nbsp;</td>
<tr>
<td>24-well plates</td>
<td>Tool</td>
<td>FALCON</td>
<td>3047</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipettes</td>
<td>Tool</td>
<td>Brand</td>
<td>747720</td>
<td>&nbsp;</td>
<tr>
<td>Forceps DUMONT #5</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors,straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>2ml; round bottom for better precipitation of pellet during centrifugation /cytospin</td>
</tr>
<tr>
<td>Cooling centrifuge</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rotation shaker</td>
<td>Tool</td>
<td>CAT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>Tool</td>
<td>Thermo Scientific</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

in vivo-like organotypic murine retinal wholemount culture

消融基因动物模型分析的目标是招收特定视网膜基因功能的经典战略。然而，转基因，基因敲除小鼠模型的视网膜特定的或有条件的往往显示早期的杀伤力或遭受严重畸形，防止超越胚胎或产后早期阶段的分析。 原代细胞培养是一种替代，以探讨外源重组的因素，在受控环境中的基因或siRNA介导的基因敲除表达的影响。游离细胞培养已减少，到达靶细胞的内源性信号的优势，从而促进确定触发后药理操纵的外生影响。但是，重要的细胞间的相互作用是最初破坏酶消化或机械分离，即使重新聚合 1 retinospheroid文化。 相比之下，器官视网膜wholemount文化提供系统在体内的情况接近的生理与神经元的相互作用和连接仍然保留 2-5 。 在这个视频文章中，我们提供了一步一步的示范（1）建立体内器官包括视网膜清扫胚胎，产后和成年鼠的眼睛（2）进行分析的分离和cytospin神经过程的特殊性wholemount文化例如器官wholemounts视网膜细胞凋亡和细胞增殖，培养后的外源重组的因素存在。

Watch this Scientific Journal Video about In vivo-like Organotypic Murine Retinal Wholemount Culture at JoVE.com

In vivo-like Organotypic Murine Retinal Wholemount Culture

此视频文章演示器官视网膜wholemount文化和cytospin一个外生的诱导效应分析程序的建立。器官型视网膜wholemount文化模仿<em&gt;在体内</em&gt;情况，同时规避经典的小鼠动物模型的缺点，大大促进了实验操作的小鼠视网膜的无障碍。

在体内的器官型小鼠视网膜Wholemount文化

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

in-vivo-like-organotypic-murine-retinal-wholemount-culture

Research

JoVE Journal

Biology

In vivo-like Organotypic Murine Retinal Wholemount Culture

17.7K 次观看.  University of Duisburg-Essen. 此视频文章演示器官视网膜wholemount文化和cytospin一个外生的诱导效应分析程序的建立。器官型视网膜wholemount文化模仿在体内情况，同时规避经典的小鼠动物模型的缺点，大大促进了实验操作的小鼠视网膜的无障碍。

视频: In vivo-like Organotypic Murine Retinal Wholemount Culture

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

器官型培养的成年兔视网膜

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

科研

生物学

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

器官型E18大鼠脑切片文化

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

全细胞记录的光在视网膜切片诱发兴奋性突触电流

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

TIRF显微镜视网膜双极细胞的影像胞外分泌

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

大鼠肝细胞的分离和小学文化

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

细胞外通量分析技术在视网膜组织外种中的能量代谢测定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

猪视网膜色素上皮细胞原代培养

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...