作者要感谢詹姆斯Oesterling流式细胞仪的帮助。这项研究是由美国国家癌症研究所（R01-CA178380，ZY）和美国国家研究院​​博士后培训补助（5T32CA009035-37，LWY）的资金支持。

已按照批准的Fox Chase癌症中心的动物护理和使用委员会的程序在执行该协议利用动物。 1，准备工具，解决方案和盖玻片<ol><li>清洁两套＃5细镊子，一组7号细弯钳，一个大的手术剪刀，1 microdissecting剪一小铲和一个带孔的勺子。将仪器的自密封消毒袋和高压锅。请眼袋仪器，直到准备使用。 </li><li>制备3％低熔点琼脂糖溶液。 <ol><li>添加1.5克琼脂糖至50毫升无菌贝科的磷酸盐缓冲液（DPBS）中，然后放入微波炉，加热至泡沫出现。漩涡烧瓶中，并再加热，直到所有的琼脂糖溶解。混合与搅拌棒的解决方案之前，如果加热团块出现。 </li><li>在37℃水浴商店解决方案，直到需要。 </li><li>对于长期存储媒体E，存储以供将来使用的琼脂糖溶液，在室温或4℃下进行几个月。再热的微波液化。在琼脂糖溶液中反复加热，加热以保持其与温暖无菌蒸馏水初始重量之前称量烧瓶中。 </li></ol></li><li>准备如前所述2木瓜蛋白酶类细胞分离通过以下方法解决: <ol><li>木瓜蛋白酶溶液，木瓜蛋白酶（100单位/毫升），半胱氨酸（0.2毫克/毫升）和DNA酶（250 U / ml）的无菌酚红含-DPBS。 </li><li>在无菌的无酚红的含DPBS，大豆胰蛋白酶抑制剂（8毫克/毫升））和DNA酶（250单位/毫升），用牛血清白蛋白的卵类粘蛋白溶液（8毫克/毫升BSA）。 过滤消毒，这两种解决方案，并调节pH值，返回酚红含的DPBS原来的颜色。 </li></ol></li><li>制备的细胞培养基（NB / B27）:补充基础培养基用1mM丙酮酸钠，2mM的L-谷氨酰胺，1％青霉素 - 链霉素（P / S）和B27补充剂。滤清器ř消毒和避光。在使用前平衡在37℃的介质和5％的CO 2。 </li><li>制备FACS缓冲液:5％的胎牛血清（FBS）的DPBS中。添加2.5毫升胎牛血清47.5毫升DPBS。 </li><li>制备聚-D-赖氨酸（PDL）包被的盖玻片上。 <ol><li>新高压灭菌盖玻片。 </li><li>外套与PDL中的盖玻片（在无菌蒸馏水中100微克/毫升）并孵育2小时，在37℃或室温下放置过夜。 </li><li>洗涤盖玻片，用无菌蒸馏水。采用NB / B27传媒洗一次。让盖玻片在使用前组织培养罩完全干燥。 </li></ol></li></ol> 2，解剖组织切片和准备<ol><li>上解剖当天，擦拭用70％乙醇和盖的吸水垫的解剖区域。 </li><li>从灭菌袋中取出解剖工具。添加冰冷，无菌DPBS / 1％P / S的小培养皿，置于冰上。 </li><li>杀头的P4 MATH1-GFP / 巢蛋白CFP小鼠的幼崽有大的手术剪刀然后按住低着头用细弯钳。取出皮肤，轻轻剥离用＃5的头骨从头骨使用刮刀细钳子然后舀出脑，并将其转移至含有DPBS / 1％P / S的培养皿中。 <ol><li>使用5号细镊子，小心地从大脑的其余部分分离小脑。一定要尽快解剖1小狗在同一时间成为可能。 </li><li>收集至少8小脑，以获得足够的非符合资格人士的分子和功能分析。 </li></ol></li><li>填充嵌入模具测量2×2×2厘米，3％低熔点琼脂糖溶液。确保琼脂糖的温度不超过37℃。 </li><li>通过在实验室组织轻轻涂抹去除小脑多余的液体。将小脑成模在垂直位置。放置在冰上的模具以允许其快速固化。使用每块〜4小脑。 </li><li>获得李咏组织切片用振动叶片vibratome。修剪含小脑，用刀片琼脂糖块。胶琼脂糖块到vibratome板与小脑定向在垂直位置。 </li><li>填补vibratome的用冰冷却的无菌DPBS / 1％P / S和地方冰下方的托盘上。 <ol><li>切成600微米的部分通过小脑的整个长度。收集使用穿孔勺子从冰盘的部分。地方的部分在培养皿中填充用DPBS / 1％P / S在冰上。 </li></ol></li></ol> 3，显微切割和细胞分离<ol><li>将含有荧光解剖显微镜下组织切片的培养皿中。 <ol><li>小心地用细镊子小脑节的其余部分由CFP +分子层和GFP +东瀛之间的夹层中（参见虚线图1）分离东瀛。小心不包括任何部分的分子层的，这将导致在污染的巢蛋白表达伯格曼胶质细胞。 </li><li>尽可能快地完成这个步骤，并保持组织在冰上，以避免细胞死亡。 </li><li>在进行下一步骤之前，除去周围的组织的琼脂糖。 </li></ol></li><li>消化使用如前所述2，得到单细胞悬浮液中的木瓜蛋白酶的基于协议的显微EGL组织。 <ol><li>重悬的细胞在FACS缓冲液用于收集通过FACS CFP-阳性细胞。 </li></ol></li></ol> 4，细胞分选及镀层<ol><li>使用无菌，高速流式细胞仪包含适当的CFP过滤器和收集细胞，流式细胞仪缓冲冰上排序细胞CFP荧光。获得每个P4小脑大约100,000非符合资格人士。 </li><li>离心细胞，在300×g离心5分钟。立即进行分子分析，或文化进行进一步的实验用的细胞。 </li><li>向培养的细胞，重新悬浮细胞在预热NB / B27米EDIA和盘上的PDL涂覆的盖玻片如前所述2。 </li></ol>

<ol>
	<li>Lee, 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=Isolation+of+neural+stem+cells+from+the+postnatal+cerebellum.">Isolation of neural stem cells from the postnatal cerebellum.</a> Nat Neurosci. 8, 723-729 (2005).</li><li>Wechsler-Reya, R. J., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+neuronal+precursor+proliferation+in+the+cerebellum+by+Sonic+Hedgehog.">Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog.</a> Neuron. 22, 103-114 (1999).</li><li>Dahmane, N., Ruizi Altaba, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+hedgehog+regulates+the+growth+and+patterning+of+the+cerebellum.">Sonic hedgehog regulates the growth and patterning of the cerebellum.</a> Development. 126, 3089-3100 (1999).</li><li>Riobo, N. A., Manning, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+signal+transduction+employed+by+vertebrate+Hedgehogs.">Pathways of signal transduction employed by vertebrate Hedgehogs.</a> Biochem J. 403, 369-379 (2007).</li><li>Dahmane, N., 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=The+Sonic+Hedgehog-Gli+pathway+regulates+dorsal+brain+growth+and+tumorigenesis.">The Sonic Hedgehog-Gli pathway regulates dorsal brain growth and tumorigenesis.</a> Development. 128, 5201-5212 (2001).</li><li>Marino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medulloblastoma:+developmental+mechanisms+out+of+control.">Medulloblastoma: developmental mechanisms out of control.</a> Trends Mol Med. 11, 17-22 (2005).</li><li>Yang, Z. J., 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=Medulloblastoma+can+be+initiated+by+deletion+of+Patched+in+lineage-restricted+progenitors+or+stem+cells.">Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.</a> Cancer Cell. 14, 135-145 (2008).</li><li>Goodrich, L. V., Milenkovic, L., Higgins, K. M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+neural+cell+fates+and+medulloblastoma+in+mouse+patched+mutants.">Altered neural cell fates and medulloblastoma in mouse patched mutants.</a> Science. 277, 1109-1113 (1997).</li><li>Li, 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=A+population+of+Nestin-expressing+progenitors+in+the+cerebellum+exhibits+increased+tumorigenicity.">A population of Nestin-expressing progenitors in the cerebellum exhibits increased tumorigenicity.</a> Nat Neurosci. 16, 1737-1744 (2013).</li><li>Ben-Arie, N., 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=Math1+is+essential+for+genesis+of+cerebellar+granule+neurons.">Math1 is essential for genesis of cerebellar granule neurons.</a> Nature. 390, 169-172 (1997).</li><li>Sutter, 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=Cerebellar+stem+cells+act+as+medulloblastoma-initiating+cells+in+a+mouse+model+and+a+neural+stem+cell+signature+characterizes+a+subset+of+human+medulloblastomas.">Cerebellar stem cells act as medulloblastoma-initiating cells in a mouse model and a neural stem cell signature characterizes a subset of human medulloblastomas.</a> Oncogene. 29, 1845-1856 (2010).</li><li>Sotelo, C., Alvarado-Mallart, R. M., Frain, M., Vernet, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+plasticity+of+adult+Bergmann+fibers+is+associated+with+radial+migration+of+grafted+Purkinje+cells.">Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.</a> J Neurosci. 14, 124-133 (1994).</li><li>Lumpkin, E. 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=Math1-driven+GFP+expression+in+the+developing+nervous+system+of+transgenic+mice.">Math1-driven GFP expression in the developing nervous system of transgenic mice.</a> Gene Expr Patterns. 3, 389-395 (2003).</li><li>Encinas, J. M., Vaahtokari, A., Enikolopov, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoxetine+targets+early+progenitor+cells+in+the+adult+brain.">Fluoxetine targets early progenitor cells in the adult brain.</a> Proc Natl Acad Sci U S A. 103, 8233-8238 (2006).</li><li>Hatten, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+regulation+of+astroglial+morphology+and+proliferation+in+vitro.">Neuronal regulation of astroglial morphology and proliferation in vitro.</a> J Cell Biol. 100, 384-396 (1985).</li><li>Lee, H. Y., Greene, L. A., Mason, C. A., Manzini, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+post-natal+mouse+cerebellar+granule+neuron+progenitor+cells+and+neurons.">Isolation and culture of post-natal mouse cerebellar granule neuron progenitor cells and neurons.</a> J Vis Exp. (23), (2009).</li><li>Emmert-Buck, M. 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=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274, 998-1001 (1996).</li><li>Espina, V., 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=Laser-capture+microdissection.">Laser-capture microdissection.</a> Nat Protoc. 1, 586-603 (2006).</li></ol>

作者什么都没有透露。

此处所描述的显微切割的方法是能够分离的活细胞中的分子和功能分析使用的纯群体的第一个过程。这表现在Li 等人9， 通过该方法得到的NEP可以培养并掺入了各种实验，并由于其高纯度的，也可用于微阵列分析。 花时间去精通这种显微切割技术，这是非常重要的。的NEP位于EGL的深区域中的非常接近的分子层，其中巢蛋白表达伯格曼胶质细胞可能污染NEP细胞的分离和妥协纯度。显微切割，因此应做得保守，以确保污染细胞在相邻的组织不包括在内。此处所描述的显微切割技术的主要优点是能够获得活细胞的能力，可培养对furtheŘ实验。由于这个原因，至关重要的是，在此过程中的所有步骤可以快速地完成，该组织被保持在冰上尽可能。此外，所有的仪器和试剂必须是无菌的。以下这些步骤将减少细胞死亡和细胞培养的污染。此外，每琼脂糖块安装多个小脑将有助于减少手术时间。 本技术不限于棉结的隔离，并且可以被用来收集来自小脑的其他领域，例如从白质分子层和神经干细胞的神经胶质细胞的细胞。旁边的小脑，显微切割可用于与包含可识别的区域，如海马，例如其他组织。使用荧光转基因的，如利用这里，可以在创建边界帮助或确定区域的夹层，其可以是在缺乏容易识​​别的区域的组织特别有用并且否则不能够是米icrodissected。因此，该技术可以帮助细胞纯化的广泛的研究领域和应用。

小脑由多个细胞层，每层含有不同的细胞类型。在开发过程中，东瀛包含增殖颗粒神经元前体（国民生产总值），而分子层和浦肯野层包含伯格曼胶质细胞和浦肯野神经元，分别。小脑深处位于脑白质，其中包含神经干细胞（NSCs）和少突胶质细胞1。 音猬蛋白（Shh的）在调节小脑发育的关键作用，特别是，它促进的GNP的增殖通过与其受体结合补丁（PTC），对Shh信号2-4的负调节物。的Shh信号传导的异常活化产生髓母细胞瘤（MB），在孩子5,6中最常见的恶性脑肿瘤。 PTC突变MB的转基因小鼠模型是MB的学习功能强大的工具，开发肿瘤类似人类MB 7,8。使用这些小鼠，它被发现的GNP是起源于刺猬型MB 7的细胞。此外，除的GNP，我们最近发现的显影小脑也可以产生MB的EG​​L内的神经元祖细胞的独特群体。这些细胞表达高水平的VI型中间丝蛋白，巢蛋白，并且被称为网络设备供应商9。的NEP位于EGL的深部内并仅在新生儿小脑发育存在短暂。他们致力于颗粒细胞系作为国民生产总值却是不同的，因为它们是静态和不表达MATH1，一个完善的标志传统国民生产总值10。此外，网络设备供应商产生MB比更有效地活化Shh信号通路9，这使得它们的新型原点为MB的肿瘤发生后的GNP。 我们以前的显微切割技术分离出小脑东瀛非符合资格人士在4（P4）日龄这里说明9。通过组织直接镜检可视显微允许小脑东瀛的具体解剖。这是必要的，因为巢是由神经干细胞在小脑白质和伯格曼胶质细胞中的分子层1,7,11,12也表示，这是至关重要的，这些细胞不被包括在内，因为他们混淆分析。细胞显微组织分离可立即用于分子分析，或者它们可以被培养用于进一步的应用。 为了有助于具体microdissecting的EGL和进一步纯化的NEP，MATH1-GFP（绿色荧光蛋白）的小鼠进行杂交以巢蛋白-CFP（青色荧光蛋白）的小鼠。转录因子MATH1通过的GNP和GFP表达特异性表达是在EGL 9,13清晰可见。 巢蛋白-CFP小鼠表达CFP的核形式，并且可以很容易地可视化的分子层9,14。总之，GFP和CFP的表达创造边界东瀛的显微切割（ 见图1）。 CFP-阳性的NEP，然后通过FACS分离，继解剖EGLS的酶解。 目前，使用最多的方法也从东瀛分离细胞是整个小脑组织15,16 Percoll密度梯度离心法。这个方法，但是，不能完全排除从分子层和白质巢蛋白+细胞群中，因此不能被用于网络设备供应商的研究。激光捕获显微切割，其使用激光能量的传递，以除去所关注的细胞，是一种用于多相组织17,18内分离特定的细胞，但捕获的细胞仅可用于DNA，RNA和蛋白质的回收和不是另一种方法能够进行培养。 该协议提供了一种专门隔离东瀛非符合资格人士的活着，纯粹的人口。这种技术也可以应用于具有任何可识别的解剖结构或荧光标记细胞/区域不同的组织类型。因此，将这一新颖显微切割技术进入细胞的分离协议的主要优点是，细胞可以从特定组织区域进行分离，以消除污染邻近的组织和细胞可以立即被收集用于分析或培养的其他应用程序。

<table border="0" cellpadding="0" cellspacing="0" width="572">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Neurobasal media</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">PDL</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">A-003-E</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:215px;">ultra pure low melting temperature agarose</td>
			<td style="width:71px;">Invitrogen</td>
			<td style="width:119px;">16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">DPBS</td>
			<td style="width:71px;">Gibco</td>
			<td align="right" style="width:119px;">14190144</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of distinct cell populations from the developing cerebellum by microdissection

显微切割是一种新技术，可以分离组织的特定区域，消除污染邻近地区的细胞来源。在这项研究是第一个使用这种方法巢蛋白表达祖商（NEP），细胞在小脑外部生发层（EGL）的新发现的种群。在组合使用显微切割用荧光激活细胞分选（FACS），棉结的纯群体是从在EGL常规颗粒神经元前体和从其它污染巢蛋白表达细胞在小脑分开收集。未经显微切割，棉结的功能分析将不会有可能与可用的电流的方法，如Percoll密度梯度离心法和激光捕获显微解剖。该技术还可以应用于用于与包含任一可识别的区域或荧光标记细胞的各种组织中的使用。最重要的是，这主要microdissectio优势Ñ​​技术是分离的细胞是活的，并且可以被培养了进一步的实验，这是目前不可能与其他所描述的方法。

如图1A所示 ，小脑切片从P4 MATH1-GFP / 巢蛋白-CFP小鼠制备的，其中，所述小脑EGL主要是由绿色荧光蛋白表达的GNP和分子层中富集由CFP-阳性的神经胶质细胞。 （; 图1A中沿虚线）以分离的NEP位于EGL的深部的，切片的EGL和分子层之间的微切割。剖分EGLS（ 图1B）然后收集用酶解随后通过FACS。 正如预期的那样，细胞在解剖东瀛的大部分（超过85％）是传统的国民生产总值，这是积极的GFP（ 图2A）。非符合资格人士（CFP +）只占5％左右的东瀛细胞群。几乎基于FACS分析无的细胞是双阳性的GFP和CFP。通过FACS纯化的CFP +细胞，进行体外培养，研究分化POTE微分方程边值问题的。 如图2B所示 ，网络设备供应商只产生了β-微管蛋白阳性神经元在培养4天后，暗示的NEP是谱系限制性神经元前体细胞。 通过这种显微协议，网络设备提供商和国民生产总值也来自东瀛的PTC不足的小脑纯化。在颅内移植，棉结开发的MB更迅速与的GNP相比，表明的NEP特别容易致癌性转化9。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52034/52034fig1highres.jpg" width="500" /> 图1标识该地区的东瀛显微切割，荧光图像从P4 MATH1-GFP /巢蛋白CFP动物（A）GFP表达坐落于东瀛，而大多数CFP表达的是位于分子层。显微切割沿吨做他黄色虚线（B）EGLS共收集组织离解。这个数字已经被修改Li 等[9]。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52034/52034fig2highres.jpg" width="500" /> 图2:网络设备供应商代表一小群细胞在东瀛，并谱系限制性神经前体细胞。 （A）流式细胞术CFP +细胞的显微EGL分离的分析（B）的NEP被分化的条件下培养4天，并染为β-微管蛋白（红色）和counterstained用DAPI（蓝色）。这一数字已经从李等人9

Watch this Scientific Journal Video about Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection at JoVE.com

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

巢蛋白表达祖细胞是在发展中国家小脑的神经元祖细胞的新发现的种群。用在这里用荧光激活细胞分选组合呈现的显微切割技术中，该细胞群可与任何其他小脑区域污染被纯化和可以培养用于进一步研究。

从开发小脑显微切割分离的不同的细胞群

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

isolation-distinct-cell-populations-from-developing-cerebellum

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.5K 次观看.  Temple University Health System. 巢蛋白表达祖细胞是在发展中国家小脑的神经元祖细胞的新发现的种群。用在这里用荧光激活细胞分选组合呈现的显微切割技术中，该细胞群可与任何其他小脑区域污染被纯化和可以培养用于进一步研究。 

视频: Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Electrophysiological Characterization of GFP-Expressing Cell Populations in the Intact Retina

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack conspicuous morphological features or show a low population density. This applies particularly to retinal amacrine cells, an exceptionally multiform class of interneurons that comprise roughly 30 subtypes in mammals1. Though being a crucial part of the visual processing by shaping the retinal output2, most of these subtypes have not been studied up to now in a functional context because encountering these cells with a recording electrode is a rare event.
Recently, a multitude of transgenic mouse lines is available that express fluorescent markers like green fluorescent protein (GFP) under the control of promoters for membrane receptors or enzymes that are specific to only a subset of neurons in a given tissue3,4. These pre-labeled cells are therefore accessible to directed microelectrode targeting under microscopic control, permitting the systematic study of their physiological properties in situ. However, excitation of fluorescent markers is accompanied by the risk of phototoxicity for the living tissue. In the retina, this approach is additionally hampered by the problem that excitation light causes appropriate stimulation of the photoreceptors, thus inflicting photopigment bleaching and transferring the retinal circuits into a light-adapted condition. These drawbacks are overcome by using infrared excitation delivered by a mode-locked laser in short pulses of the femtosecond range. Two-photon excitation provides energy sufficient for fluorophore excitation and at the same time restricts the excitation to a small tissue volume minimizing the hazards of photodamage5. Also, it leaves the retina responsive to visual stimuli since infrared light (&gt;850 nm) is only poorly absorbed by photopigments6.
In this article we demonstrate the use of a transgenic mouse retina to attain electrophysiological in situ recordings from GFP-expressing cells that are visually targeted by two-photon excitation. The retina is prepared and maintained in darkness and can be subjected to optical stimuli which are projected through the condenser of the microscope (Figure 1). Patch-clamp recording of light responses can be combined with dye filling to reveal the morphology and to check for gap junction-mediated dye coupling to neighboring cells, so that the target cell can by studied on different experimental levels.

This article depicts the recording of individual cells from fluorescently tagged neuronal populations in the intact mouse retina. By using two-photon infrared excitation transgenetically labeled cells were targeted for patch-clamp recording to study their light responses, receptive field properties, and morphology.

在完整的视网膜电生理特性表达GFP的细胞群

Studying the physiological properties and synaptic connections of specific neurons in the intact tissue is a challenge for those cells that lack ...

科研

神经科学

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

激光捕获显微切割的丰富种群的创伤性脑损伤后神经元或单个神经元的基因表达分析

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Genetic modification of specific regions of the developing mammalian brain is a very powerful experimental approach. However, generating novel mouse mutants is often frustratingly slow. It has been shown that access to the mouse brain developing in utero with reasonable post-operatory survival is possible. Still, results with this procedure have been reported almost exclusively for the most superficial and easily accessible part of the developing brain, i.e. the cortex. The thalamus, a narrower and more medial region, has proven more difficult to target. Transfection into deeper nuclei, especially those of the hypothalamus, is perhaps the most challenging and therefore very few results have been reported. Here we demonstrate a procedure to target the entire hypothalamic neuroepithelium or part of it (hypothalamic regions) for transfection through electroporation. The keys to our approach are longer narcosis times, injection in the third ventricle, and appropriate kind and positioning of the electrodes. Additionally, we show results of targeting and subsequent histological analysis of the most recessed hypothalamic nucleus, the mammillary body.

Genetic Manipulation of the Mouse Developing Hypothalamus through In utero Electroporation

Despite the functional and medical importance of the hypothalamus, in utero genetic manipulation of its development has rarely been attempted. We show a detailed procedure for in utero electroporation into the mouse hypothalamus and show representative results of total and partial (regional) hypothalamic transfection.

遗传操纵鼠标的发展下丘脑通过<em&gt;在子宫内</em&gt;电穿孔

Genetic  modification of specific regions of the developing mammalian brain is a very  powerful experimental approach. However, generating novel mouse ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

通过激光捕获微分路，从小鼠脊髓运动神经细胞体中生成RNA进行转录分析

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

有丝分裂中的不同发育阶段小鼠胚胎皮层实时成像

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

一种从人类死后小脑中的激光捕获微解剖 purkinje 细胞中分离高质量 rna 的不锈钢协议

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...