Supported by National Institutes of Health (DK55679, and DK59888 to A.N.). Emory University&#160;Integrated Cellular Imaging Microscopy Core of the Winship Cancer&#160;Institute comprehensive cancer center grant, P30CA138292, and the Crohn's and Colitis Foundation of America Career Development award to C.T.C. and Fellowship Award to A.E.F.

实验中使用的10和12周龄之间C57BL / 6小鼠。使用动物的所有程序进行了审查并批准了埃默里大学机构动物护理和使用委员会，并根据卫生标准的国家机构进行。 1.实验装置<ol><li>建立了低温恒温器<ol><li>平衡低温恒温器到-20℃，其中包括刀片切片机卡盘和（行使围绕叶片格外小心！）。 </li><li>填充OCT空cryomold（最佳切削温度化合物）和平衡至-20℃（〜30分钟）。从模具中取出冷冻OCT和修复它与液体华侨城夹头。把块/夹头的手臂切片机，让华侨城为10分钟设置。 </li><li>将恒温器每节10微米，刮胡子华侨城块，直到它是平的。几厘米备份切片臂远离刀片。此时不调整刀片或夹头的的EXPER剩余iment。 </li></ol></li><li>准备刀片:准备每个样品2刀片。使用金​​属文件，沉闷叶片的边缘sharped。接下来，去除铝法兰用钳子。 </li><li>预冷一个耐热盘或其他平面上的干冰。 </li><li>准备一个夹层菜10-15解剖针。将10cm组织培养皿50％填充硅就足够了。加入5毫升Hanks缓冲液在室温下。 </li></ol> 2.解剖远端结肠组织<ol><li>小鼠放置在一个密封的腔和安乐死小鼠吸入异氟醚和颈椎脱位，再喷70％的乙醇到腹部和胸部12。 </li><li>做一个小切口，用剪刀腹部皮肤，然后做一个切口暴露腹腔。类似的程序，可以在这里找到12描述。 </li><li>切大肠在肛缘和捉弄除了肠系膜，直到大肠是免费的。 </li><li>切除远端结肠0和6厘米的肛门之间的段。这里，隐窝深度为〜150微米（参见图1B）。纵向切开用剪刀打开结肠和删除粪内容。 </li><li>针组织的解剖菜粘膜表面朝上。伸展在Hanks缓冲硅菜用组织解剖针，让休息在室温（ 图1C）10分钟。这消除褶皱从组织，并允许肌肉层放松。 </li></ol>在低温恒温器用于切片3.安装组织<ol><li>滑动刀片下组织中所需的部分，然后倒出汉克斯缓冲。添加另一个刀片顶端，制作三明治。切割组织段和取出针。剃刀刀片/组织三明治转移到干冰。允许组织冻结（5-10分钟， 图1D）。 </li><li>拿起剃刀/组织三明治，并允许它通过将手指在顶部BL略暖ADE（粘膜面朝上。此定向的组织，以使基底肌肉层的第一切片。）。分离三明治和切割组织周围部分的边缘。切割组织段大约0.5厘米×1厘米。边缘区域有一种倾向，卷曲，造成连续切片包含许多组织层。请注意，过切比下切好，所以宁可谨慎的一面。 </li><li>允许组织温热直到在组织上的顶部的冰开始融化。在这一点上迅速小心地按入组织在低温恒温器华侨城块。 </li><li>通过将手指在样品取出刀片。热传递将让你卸下刀片而不破坏组织。 </li><li>大衣的组织与华侨城和平衡5分钟。切段在10微米（ 图1E中的F）。目视检查部分，直到墓穴被认为。地方的部分在1.5ml管或上滑动。 </li><li>对于基因或蛋白质，地方的t分析他样品管中5地穴基地，5个过渡性的，每管5表面部分。对于RNA集合加入1 ml商业隔离剂和在室温下孵育10分钟。根据制造商的说明进行RNA纯化。使用5-10微克总RNA的基因产生。 </li><li>蛋白质纯化，增加0.5冷马国贤毫升裂解每5个部分缓冲（加蛋白酶抑制剂）。超声处理3次，在70％的周期，在冰上。 <ol><li>添加样品进行SDS PAGE上样缓冲液（Laemmli缓冲液），并孵育在100℃下进行10分钟。受试者样品进行SDS PAGE和转移，然后在5％牛奶/ PBS阻断2小时。进行免疫印迹分析随后孵化与KLF4抗体（1:500）O / N在4℃。 </li></ol></li></ol>

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The authors have nothing to disclose

涉及在结肠上皮细胞增殖和分化的分子机制知之甚少。这包括在我们的干细胞区室中在结肠上皮隐窝的底部的细胞信号环境的了解差距。也有在背后肠分化过程的兴趣日益增​​加。例如， 增加体内分化的认识，需要为基准，进行研究，以在体外生产初级肠道系统16。 上述过程包含了需要特别注意几个步骤。首先，去除冷冻组织段周围的边缘（如3.2节中讨论）是必需的组织边缘解剖和冷冻过程中卷曲。未按动这些边缘的结果是表面和隐窝基底细胞的混合群。安装预冷，分级华侨城块的组织也是必不可少的。 Ť他的协议可以通过改变在每个池的部分的数量适应于远端结肠的其他区域。例如， 如图1B所示 ，150-300微米之间的粘膜隐窝长度变化。因此，从肛门学习4厘米-6厘米之间的区域时，这些区段的数目应从15增加到30。重要的是，该协议不建议用于近端结肠（7厘米9cm）中，由于褶皱（皱襞obliquae），其延伸进入所述内腔因此当单独表面和隐窝基底细胞通过连续切片是不可能的。 连续切片技术已被用于研究隐窝基和在人体内的表面上皮细胞群。然而，我们的协议增加了需要由于鼠组织的小尺寸的附加步骤。我们建议，上述协议将允许隐窝基和表面的上皮细胞群体在遗传听话鼠标系统的调查。事实上，汇集部分一愕LD优质蛋白质和RNA适合下游分析。未来的应用可能包括细胞信号传导途径或染色质免疫沉淀的研究。但是，应该指出的是，像几个上述的替代方法中，将所得切片包含上皮细胞和间质固有层细胞的混合物。总之，这里描述的方案提供了一种快速，高产率方法对分子过程的在小鼠结肠黏膜的空间上不同的区域进行分析。

结肠上皮衬里是一种高度再生的组织，与驻地干细胞补充组织每隔几天1。再生的这个过程需要增殖干细胞区室的维护。随着时间的推移，新产生的子细胞失去增殖潜能和迁移朝小肠的腔表面。暗合了迁移，祖细胞分化为成熟的阻隔形成肠或粘膜产生杯状细胞。这种分化方案的失败被认为有助于受损上皮屏障诸如炎症性肠病和结肠癌2,3是观察。 上述过程的详细研究，需要方法用于分离的隐窝基和表面的细胞群。多种方法都存在，各有独特的优点和缺点。目前的方法为肠上皮细胞的分离包括沟道elating剂和机械解离，导致整个隐窝，上皮片，或单细胞的，依赖于实验条件4,5的分离。这些都是高产的方法，但在变化间信号已经在这些条件，可能无法代表本土环境下6,7报道。激光捕获显微切割允许空间不同材料的集合，但实现了低分子量收率8,9。在人结肠组织，序列水平cryosectioning方法已经发展10。然而，小鼠的粘膜组织有小望而却步这种技术的直接拨款。在人类中，肠隐窝长度在结肠（隐窝基和表面细胞之间的遥远）之间变化100-1,000〜11微米。在小鼠中，隐窝长度之间〜50-300微米（ 见图1A）。鼠隐窝的小尺寸提出了挑战伊索拉化利用现有的协议这两种细胞群。 现在我们描述一种低成本，高产量的方法的隐窝基底的分离和在小鼠结肠组织表面的上皮细胞群。组织收获以这种方式然后可通过许多标准的下游应用，包括免疫荧光染色，RT-PCR（实时聚合酶链式反应）或蛋白质印迹进行分析。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems, Nussloch Germany</td>
			<td>CM 1510-3</td>
			<td></td>
		</tr>
		<tr>
			<td>MyiQ real time PCR detector</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM 510&#160;</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Tissue-Tek, Sakura Finetek, Torrance CA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>cryo-mold</td>
			<td>Tissue-Tek, Sakura Finetek, Torrance CA</td>
			<td>4557</td>
			<td>25mmx20mmx5mm</td>
		</tr>
		<tr>
			<td>High profile microtome blade</td>
			<td>Leica Biosystems, Nussloch germany</td>
			<td>818</td>
			<td></td>
		</tr>
		<tr>
			<td>GEM industrial blades</td>
			<td>American Safety Razor Blades</td>
			<td>62-0314</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard silicon elastomere base</td>
			<td>Dow Corning, Midland MI</td>
			<td>3097358</td>
			<td></td>
		</tr>
		<tr>
			<td>27 1/2 g needle</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>305109</td>
			<td>pins for dissection</td>
		</tr>
		<tr>
			<td>TRizol</td>
			<td>Life Technologies</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Rneasy</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody ZO-1</td>
			<td>Invitrogen&#160;</td>
			<td>187430</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody KLF4</td>
			<td>Cell Signaling</td>
			<td>4038S</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody mGAPDH</td>
			<td>Sigma</td>
			<td>G8795</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody Secondary Alexa conjugate</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>488 anti-rabbit</td>
		</tr>
		<tr>
			<td>Antibody Secondary HRP conjugate</td>
			<td>Jackson</td>
			<td>111/115-001-003</td>
			<td>rabbit/mouse&#160;</td>
		</tr>
		<tr>
			<td>Topro-3</td>
			<td>Invitrogen</td>
			<td>T3605</td>
			<td></td>
		</tr>
		<tr>
			<td>HANKs balanced salt buffer</td>
			<td>Life Technologies</td>
			<td>14025092</td>
			<td></td>
		</tr>
		<tr>
			<td>RIPA lysis buffer</td>
			<td></td>
			<td></td>
			<td>50mM Tris-HCl pH 7.4 150mM NaCl 1% NP40 0.25% Na-deoxycholate</td>
		</tr>
		<tr>
			<td>PCR Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>primer TBP 3&#39;</td>
			<td></td>
			<td></td>
			<td>GGAATTGTACCGCAGCTTCAAA</td>
		</tr>
		<tr>
			<td>primer TBP 5&#39;</td>
			<td></td>
			<td></td>
			<td>GATGACTGCAGCAAATCGCTT</td>
		</tr>
		<tr>
			<td>primer KLF4 3&#39;</td>
			<td></td>
			<td></td>
			<td>TGTGACTATGCAGGCTGTGGCAAA</td>
		</tr>
		<tr>
			<td>primer KLF4 5&#39;</td>
			<td></td>
			<td></td>
			<td>ACAGTGGTAAGGTTTCTCGCCTGT</td>
		</tr>
	</tbody>
</table>

cryosectioning method for microdissection of murine colonic mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

结肠组织切片进行组织学评估和H＆E染色13。粘膜的传统截面图被认为是在图2A中 。基底区域包含了外肌层，主要的圆形组成肌层。继续向着内腔，粘膜肌层是显而易见邻近于隐窝基底上皮细胞，移行细胞是在组织的中间，最后，表面细胞群体面对肠腔。这里所描述的连续切片方法保持的取向使得纵向和圆形肌层首先遇到（ 图2B），随后粘膜肌层（ 图2C）。注意非肌肉间质细胞中的图像的左上角的外观。对于下面的下游分析所述，肌肉部分被丢弃。后续章节包含上皮细胞和间质组织（固有层），开始与隐窝基细胞（ 图2D）。注意缺乏在大多数隐窝，从而出现较暗的中央管腔由于紧密堆积细胞核。这些层包含增殖车厢。过渡单元出现排列紧密的腺体与著名的中央流明（ 图2E）。最后，表面的细胞群具有不规则结构，与上皮单层观看连接面 （ 图2F）的区域。 如上所述连续切片也可用于免疫荧光标记和共聚焦显微镜处理（如这里描述14）。 图2G示出了分离的隐窝固定和透化在100％甲醇中且随后染色细胞核（蓝色）和细胞-细胞连接蛋白小带Occludens 1（ZO-1，绿色）。在传统的切片方向，隐窝和表面细胞可以同时观看（ 图2G </strong&gt;的）。注意，ZO-1交界污点行结肠隐窝的内腔中。管腔此Z0-1衬里也可以看出移行细胞群的串行截面样品中，在圆形的腺体（ 图2G）的中心。增强ZO-1染色可见在表面的细胞群（ 图2I和 J）。 几个分化因子已知是表示在沿着隐窝 - 面轴时空方式。这包括转录因子Kruppel-样因子4（KLF4），这是已知的表面的细胞群进行富集。使用传统方法切片，KLF4在面对表面的细胞群（ 图2K）流明的细胞核中。因此，我们推测，KLF4的mRNA将主要表现在表面的细胞群。为了测试这一点，连续切片被收集并分为表面，移行和隐窝基样品。 Subsequently，RNA使用标准的Trizol提取收获，用一个额外的纯化的RNeasy柱和DNA酶处理。 RNA从5汇集连续部分，每个样品的总RNA之间的5-10微克这产生了回收。然后将这些样品进行半定量实时PCR为KLF4和参照基因，TATA盒结合蛋白1（TBP-1）（ 图2L）14。如图2L，正常化与TBP-1后，发现表面细胞以表达高达8倍的KLF4基因被表达在隐窝细胞。同样，KLF4蛋白水平被认为具有调节空间沿隐窝面轴。如在4℃下如上所述，然后温育在裂解缓冲液20分钟连续切片汇集。这产生200-250微克每个样品的蛋白质之间。然后这些样品进行分析通过SDS-PAGE（ 图2M）15。如图2M，KLF4 protein水平显着表面的细胞群高。上述结果证明这种协议的孤立大量的表面和隐窝上皮细胞从小鼠结肠黏膜的能力。 <img alt="图1" src="/files/ftp_upload/53112/53112fig1.jpg" /> 图1:（A）示意图，显示小鼠结肠黏膜和外部的肌肉层。我们的方法旨在通过串行cryosectioning（每10微米），收集分散的表面细胞和隐窝基细胞群。外部肌肉层丢弃。 （B）的隐窝高度沿结肠（隐窝基和表面层之间的距离）的长度而改变。数据点表示个人隐窝测量和符号表示生物学重复（N = 4）。 （C）段结肠收集，一分为二，并固定到一个硅解剖盘。 （D）一个组织本身gment从肛门约3cm时之间按压剃刀刀片，在干冰上冷冻。 （ 五）组织，然后安装预调平华侨城块。 （F）冷冻切片被删除并目视检查。 <img alt="图2" src="/files/ftp_upload/53112/53112fig2.jpg" /> 图2:代表性数据 （A） 的结肠组织中的横截面用苏木精-曙红显示圆肌层（厘米），粘膜肌层（毫米），隐窝基底细胞，移行细胞，以及表面的细胞。 （B - C）结肠肌肉层。 （D）地穴基细胞。注意没有中央内腔。 （ 五）过渡细胞。 （F）表面的细胞。 （G）免疫荧光染色的孤立结肠隐窝。 ZO-1（绿色）和细胞核（蓝色）的横截面观察。 （HI）ZO-1和过渡和表层细胞细胞核染色。 （J）的ZO-1染色放大图。 （K）的KLF4（绿色）富含表面的细胞群。 （L）上的三个室之间KLF4 mRNA水平的实时PCR评估。 （M），KLF4蛋白水平在这些细胞群体的Western印迹分析。

Watch this Scientific Journal Video about Cryosectioning Method for Microdissection of Murine Colonic Mucosa at JoVE.com

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

Cryosectioning方法小鼠结肠黏膜显微切割

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

cryosectioning-method-for-microdissection-of-murine-colonic-mucosa

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

17.3K 次观看.  Emory University, Atlanta, Georgia. Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

视频: Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Flash Freezing and Cryosectioning E12.5 Mouse Brain

Demonstrated in this video are the techniques for flash freezing and sectioning embryonic brain tissue from mouse. Useful tips for using the cryostat are given, including troubleshooting methods that can be used while cutting to ensure that the resultant tissues sections are free of cracks and other distortions.

闪存冻结和Cryosectioning E12.5小鼠脑

科研

生物学

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

激光捕获显微切割哺乳动物组织

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

显微切割的黑寡妇蜘蛛丝绸生产腺体

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

文化与偏振刺激人体肠道黏膜植的新方法

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

检查荧光模式Cryosectioning酵母社区

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

鼠阀门内皮细胞的分离

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

激光辅助显微切割（LAM）作为个体细胞类型的转录谱的工具

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

A Morphometric and Cellular Analysis Method for the Murine Mandibular Condyle

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the structural and cellular components of the mandibular condylar cartilage (MCC). This manuscript describes methods for analyzing these changes and a method for altering the loading of the TMJ in mice (i.e., compressive static TMJ loading). The structural evaluation illustrated here is a simple morphometric approach that uses the Digimizer software and is performed in radiographs of small bones. In addition, the analysis of cellular changes leading to alterations in collagen expression, bone remodeling, cell division, and proteoglycan distribution in the MCC is described. The quantification of these changes in histological sections - by counting the positive fluorescent pixels using image software and measuring the distance mapping and stained area with Digimizer - is also demonstrated. The methods shown here are not limited to the murine TMJ, but could be used on additional bones of small experimental animals and in other regions of endochondral ossification.

This manuscript presents methods for analyzing morphometric and cellular changes within the mandibular condyle of rodents.

小鼠下颌骨髁突的形态学及细胞分析方法

The temporomandibular joint (TMJ) has the capacity to adapt to external stimuli, and loading changes can affect the position of condyles, as well as the ...