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><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&amp;nbsp;</td><td>Carl Zeiss</td><td></td><td>Confocal microscope</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>Antibody ZO-1</td><td>Invitrogen&amp;nbsp;</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&amp;nbsp;</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>primer TBP 3&#039;</td><td></td><td></td><td>GGAATTGTACCGCAGCTTCAAA</td></tr><tr><td>primer TBP 5&#039;</td><td></td><td></td><td>GATGACTGCAGCAAATCGCTT</td></tr><tr><td>primer KLF4 3&#039;</td><td></td><td></td><td>TGTGACTATGCAGGCTGTGGCAAA</td></tr><tr><td>primer KLF4 5&#039;</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

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

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

隔离和免疫淋巴细胞和树突状细胞的小鼠Peyer氏斑

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

科研

免疫与感染

Using Robotic Systems to Process and Embed Colonic Murine Samples for Histological Analyses

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of experimental models needs to be as rigorous as that applied for human samples. Indeed, drawing reliable and accurate conclusions is critically influenced by the quality of tissue section preparation. Here, we describe a protocol for histopathological analysis of murine tissues that implements several automated steps during the procedure, from the initial preparation to the paraffin embedding of the murine samples. The reduction of methodological variables through rigorous protocol standardization from automated procedures contributes to increased overall reliability of murine pathological analysis. Specifically, this protocol describes the utilization of automated processing and embedding robotic systems, routinely used for the tissue processing and paraffin embedding of human samples, to process murine specimens of intestinal inflammation. We conclude that the reliability of histopathological examination of murine tissues is significantly increased upon introduction of standardized and automated techniques.

Lack of standardization for murine tissue processing reduces the quality of murine histopathological analysis as compared to human specimens. Here, we present a protocol to perform histopathological examination of murine inflamed and uninflamed colonic tissues to show the feasibility of robotic systems routinely used for processing and embedding&#160;human samples.

利用机器人系统对结肠小鼠样本进行处理和嵌入进行组织学分析

The understanding of human diseases has been greatly expanded thanks to the study of animal models. Nonetheless, histopathological evaluation of ...

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 ...

神经科学

Performing Colonoscopic-Guided Pinch Biopsies in Mice and Evaluating Subsequent Tissue Changes

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount importance when studying diseases of the gastrointestinal (GI) tract. The murine colonic pinch biopsy model is a useful tool to define these processes. Additionally, the interplay between gut luminal content (e.g., microbes) and the colon can be studied. However, wound induction and the ability to track wound closure over time in a reliable manner can be challenging. Moreover, tissue preparation and orientation must be carried out in a standardized way to optimally interrogate histologic and molecular changes. Here, we present a detailed method describing biopsy-induced injury and the monitoring of wound closure through repeat colonoscopies. An approach is described that ensures consistent and reproducible measurements of wound size, the ability to collect the wound bed for molecular analyses as well as visualize the wound bed upon sectioning of tissues. The ability to successfully carry out these techniques allows for studies of the acute injury response, wound healing and luminal-host interactions within the colon.

Here, we provide a detailed description of the procedure to induce colonoscopic-guided pinch biopsies in mice and track wound closure in real time. Additionally, methods for the preparation of tissues for histological, immunohistochemical and molecular analyses of the wound bed are provided.

在小鼠中进行结肠镜引导捏活检并评估后续组织变化

Understanding the tissue and cellular changes that occur in the acute injury response as well as during the wound healing process is of paramount ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

从小鼠和人类结肠组织初级肌成纤维细胞的分离

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

生物学

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 ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

原代培养小鼠肠嗜铬细胞的全细胞电生理

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Snap Freezing of Muscle Tissue for Sectioning: A Protocol to Obtain Ultrathin Sections of Rapidly Frozen Murine Muscle Tissue

Source: Bonetto, A. et al. <a href="https://www.jove.com/video/54893" target="_blank">The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia.</a> J. Vis. Exp. (2016)
This video describes the procedure for snap freezing muscle tissue and obtaining its thin sections. The technique can help study cancer-related cachexia in colon cancer mouse models.

用于切片的肌肉组织快速冷冻:获得快速冷冻小鼠肌肉组织超薄切片的方案

Source: Bonetto, A. et al. The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia. J. Vis. Exp. (2016)
This video ...

JoVE Encyclopedia of Experiments

癌症研究

Gastrointestinal Cancer

Animal model studies

Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model

Source: Schewe, M. et al. <a href="https://www.jove.com/video/56329" target="_blank">The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a technique to isolate whole crypts from a mouse&#8217;s small intestine. The isolated whole crypts can be used to propagate long-lived and self-renewing 3D organoids in vitro.

肠隐窝分离:一种从小鼠模型小肠中分离整个隐窝的方法

Source: Schewe, M. et al. The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells. J. Vis. Exp. (2017)
In ...

Frozen Mouse Brain Tissue Sectioning: A Procedure to Obtain Thin Frozen Tissue Sections from Frozen Murine Brain Tissue

Source: Comba, A. et al. <a href="https://www.jove.com/v/60939/laser-capture-microdissection-glioma-subregions-for-spatial-molecular">Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion.</a> J. Vis. Exp. (2020)
In this video, we demonstrate the sectioning of a frozen mouse brain tumor tissue using a cryostat. The frozen brain tissue sections so obtained are stored at low temperatures until further analysis.

冷冻小鼠脑组织切片:从冷冻小鼠脑组织获得薄冷冻组织切片的程序

Source: Comba, A. et al. Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, ...