We would like to thank Ainara Ruiz de Sabando for providing H&amp;E images. This work was supported by grants from the National Institutes of Health (DK052230, DK093680 and CA172113) awarded to Dr. Vincent W. Yang.

1.小鼠<ol><li>所有涉及小鼠的研究由纽约州立石溪大学机构动物护理和使用委员会（IACUC）批准。将小鼠保持在12:12小时光照 - 黑暗周期。 <ol><li>商购获得C57BL / 6小鼠。获得C57BL / 6小鼠的携带loxP位点侧翼KLF5等位基因（KLF5˚FL / F 1）。这些小鼠先前描述的21和永井良三博士慷慨地提供。 </li></ol></li><li>购买携带下LGR5子（LGR5-EGFP / CreERT2小鼠）的调节可诱导Cre重组基因C57BL / 6小鼠。 </li><li>要建立LGR5-EGFP / CreER T2 / KLF5 F 左 / F 左小鼠，越过KLF5 FL / F 左小鼠LGR5-EGFP / CreER T2小鼠，然后回交后代为KLF5 FL / F 左 </SUP&gt; 老鼠。 </li><li>他莫昔芬按照协议对待老鼠前面所述22。注入8周龄的实验和对照小鼠腹膜内（IP）用1mg他莫昔芬（10毫克/毫升），溶解于无菌玉米油，连续5天。 </li><li>在第一次注射他莫昔芬后第14天，通过将它们放置在连接到CO 2气体源的腔牺牲使用二氧化碳窒息的小鼠。允许气体流入腔室，直到小鼠是无意识和所有运动停止。为了确保执行安乐死颈椎脱位。 </li></ol> 2.组织准备和瑞士的滚动<ol><li>在腹部侧的皮肤用解剖镊子和剪刀，切一个切口。用一对镊子，保持在切口皮肤和拉从腹部肌肉组织轻轻的路程。用剪刀切开腹部皮肤和暴露腹肌。 </li><li>保持和拉起PE用钳子ritoneum。确保不要举行，并在肠子拉。精心制作的腹膜组织的切口，并继续切开皮肤，露出腹部肌肉。仔细使腹部肌肉的切口，并继续削减他们掉，露出肠子。 </li><li>确定在结肠和跟踪到的远端，它加入直肠/肛门。用剪刀，切割尽量靠近肛门开口越好。 </li><li>识别胃和跟踪在其与小肠的联接。凭借一把剪刀，剪释放肠从胃约1厘米的距离。被释放的小肠和结肠的整个长度传送到含有磷酸盐缓冲盐水（PBS）一个干净的培养皿。 </li><li>用一只手或一对钳子的，持有结肠的远端，并使用另一只手轻轻地从任何肠系膜结缔组织和/或脂肪组织解开结肠的整个长度。 </li><li>确定盲肠（小肠和大肠之间的小囊），并切其中盲肠和结肠加入（结肠的近端）以释放结肠。 </li><li>用一只手或一对镊子，保持小肠，并使用另一只手轻轻地从任何肠系膜结缔组织和/或脂肪组织解开小肠的整个长度。切去盲肠和丢弃，如果没有必要的。 注意:该方法可以在这里通过孵育在改性布安的固定液的解剖出肠（50％乙醇/ 5％乙酸在卫生署2 O）被停止过夜，并继续该过程翌日。 </li><li>处理肠（小肠或结肠）中的一个环节，填充10毫升注射器修改布安固定液及附加灌胃针吧。插入针头有关在肠段的前开口半厘米。 </li><li>通过应用公司的压力与仪器握住肠段内灌胃针对肠道段。用另一只手握住注射器，施加轻柔但一致的压力冲洗使用改性布安固定液肠道段的内容。这个步骤可以使肠段和直接固定清洁同步。使用培养皿收集流过的浪费。固定可以通过结肠颜色转动不透明被观察到。 注意:确保不冲洗或小肠段可能会爆开时施加太大的压力。 </li><li>用剪刀剪沿肠系膜线呈纵向打开肠段。用钳子捏住，然后在含有PBS培养皿简单冲洗。 <ol><li>切小肠分为3段:近，中，和远端。近侧段是以下胃所述一个立即，和远端段是冒号之前紧接之一。近侧段相当于十二指肠，月中的空肠，并在远端回肠。 </li><li>对于每个段标记近端和远端。近端是最初朝向胃指向所述一个，并朝向结肠指出所述远端。 </li></ol></li><li>使用培养皿的顶部放置清洗和开肠段。放置肠段与腔侧朝上。 <ol><li>对于结肠，通过在其宽度运行variegations /脊识别腔侧和存在于近端。中期和远端地区有纵向运行一些variegations。对于小肠，由粗显现表面，这标志着绒毛识别管腔侧。 注意:对于小肠，有近，中，远地区没有外部宏观解剖划分。然而，这些区域可以在显微镜下章节中可识别。绒毛是在近端时间最长的地区，并成为逐渐变短远端他们在哪里最短的长度。显微镜下，结肠隐窝是其中也可以通过脊的存在所确定的近端区域最短的。结肠隐窝是在中段的远端区域最高和更短，但仍比在近端区域高。 </li></ol></li><li>与瑞士滚动结肠进行: <ol><li>保持结肠平开，并与朝培养皿的边缘的近端钳拉出。 </li><li>保持腔侧朝上，并与用一只手钳的近端并用另一只手握住一根牙签。 </li><li>使用镊子环绕牙签近端的边缘，稍微捏边包裹着对牙签固定到位。 </li><li>轻轻地，慢慢地开始滚动用手指牙签打滚牙签结肠，形成一个瑞士卷。 </li><li>一旦整个结肠长度已经卷起，用一对镊子小心地滑的C敖瑞滚下牙签进入组织处理/ -embedding磁带。放置在10％缓冲福尔马林盒在室温下过夜。 </li></ol></li><li>与瑞士滚动小肠进行: <ol><li>一次处理一个段，保持段平与腔侧开放并与来自朝向培养皿的边缘的近端钳子拉出。 </li><li>瑞士继续滚动作为结肠描述。 </li></ol></li><li>随着加工福尔马林固定组织的石蜡包埋继续。步骤如下: 注:使用一个自动处理器（请参阅材料和设备的详情）。 <ol><li>尽管组织是在纸盒，冲洗组织与PBS固定液，直到完全去除。 </li><li>按以下顺序用乙醇脱水组织:50％乙醇10分钟，70％乙醇10分钟，80％乙醇10分钟，95％乙醇10分钟，100％乙醇10分钟，100％乙醇10分钟和1-00％乙醇10分钟。 </li><li>交换乙醇用二甲苯按以下顺序:2:1乙醇:二甲苯10 - 15分钟，1:1乙醇:二甲苯10 - 15分钟，1:2乙醇:二甲苯10 - 15分钟，100％二甲苯10 - 15分钟，100％二甲苯10 - 15分钟，并进行10 100％二甲苯 - 15分钟。注意:二甲苯是一种健康危害，因此工作场所应配备有局部排气通风，适当罩和适当的防护设备应该由人员来穿。 </li><li>交易所二甲苯石蜡。请在54炉设置一个真空下列步骤: - 58℃:2:1的二甲苯:石蜡30分钟，1:1的二甲苯:石蜡30分钟，1:2的二甲苯:石蜡为30分钟，100％石蜡为1 - 2小时，进行1 100％石蜡 - 2小时或过夜。 注意:不要让石蜡超过60℃的长时间，因为这会降低石蜡聚合物，使其硬而脆。 </li><li>嵌入全新的石蜡和东方组织的德石蜡硬化之前sired。对于瑞士卷，下列组织石蜡化，它石蜡包埋过程中重新定位在其一侧每卷是非常重要的。这确保了在切割过程中，各轧辊的整个长度的部分会产生。 </li></ol></li><li>对于染色，用切片机切成5微米厚的切片。收集带电的幻灯片的章节，并在65℃干燥它们（烘焙）烘箱过夜放冷至室温，然后用它们进行染色。 注:焙烧时间可以缩短到1 - 取决于滑动如何新鲜切割是2小时。如果不到，因为切片一个月，然后最好是烘烤过夜。如果已经一个多月了，那么1 - 烘烤2小时就足够了，以节省时间。如果时间不多了，可以随时使用一夜之间后盾。 </li></ol> 3.组织学分析和免疫荧光染色进行组织学分析和immunofluorescence染色如前所述23,24，与修改。对于增强型绿色荧光蛋白（EGFP）免疫荧光: <ol><li>烘烤在65℃的烘箱载玻片1小时以失活的内源性碱性磷酸酶活性，并随后在二甲苯deparaffinize。 </li><li>孵育切片中的甲醇，以阻断内源性组织过氧化物酶3％过氧化氢的浴;然后通过温育在随后在柠檬酸盐缓冲溶液的抗原修复递减醇浴系列（100％，95％，70％）在125℃再水合（10mM柠檬酸钠，0.05％吐温-20，pH 6.0）中10分钟使用decloaking室。 </li><li>绘制围绕组织切片的圆用标记笔，提供疏水阻挡层和在37℃（（在TBS-吐温2.5％牛血清白蛋白〔BSA〕）封闭缓冲液30分钟，然后用第一抗体针对EGFP稀释1孵育切片:500），在4℃下在加湿室中过夜，同时轻轻振动。</li><li>洗部分，并在37℃下在适当的浓度与荧光标记的第二抗体孵育30分钟。 </li><li>用Hoechst二级抗体治疗和细胞核染色后进行清洗的幻灯片，并与安装装入介质。 <ol><li>对于KLF5 / EGFP / Ki-67的联合染色过程幻灯片按照步骤3.1中所述 - 3.3通过与KLF5抗体（稀释1:150）孵育执行KLF5染色过夜。 </li><li>应用兔AP聚合物检测，以幻灯片按协议。 </li><li>显示使用显色免疫组化染色按照制造商的协议Klf4的染色。 </li><li>执行使用先前描述的方案25抗体溶出。在50℃下在甘氨酸SDS（pH为2.0）在搅拌1小时溶液孵育载玻片。 </li><li>洗涤用蒸馏水彻底滑动并在37℃下进行30分钟（在TBS-吐温2.5％BSA）封闭缓冲液孵育。 </li><li>添加Ki-67的（稀释1:500）和EGFP（稀释至1:500）同时抗体并在37℃下孵育1小时。 </li><li>用Hoechst染色（数据未显示）之前，执行适当的二级抗体荧光检测和安装介质装入。 </li></ol></li><li>观察染色用苏木伊红（H＆E）26 5微米的部分组织的组织形态。对于荧光图像，使用的535，646和700发射波长分别以可视化EGFP，KLF5和Ki-67染色。 </li></ol>

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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kruppel-like+factor+5+is+important+for+maintenance+of+crypt+architecture+and+barrier+function+in+mouse+intestine.">Kruppel-like factor 5 is important for maintenance of crypt architecture and barrier function in mouse intestine.</a> Gastroenterology. 141 (4), 1302-1313 (2011).</li><li>Nandan, M. O., 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=Kruppel-like+factor+5+is+a+crucial+mediator+of+intestinal+tumorigenesis+in+mice+harboring+combined+ApcMin+and+KRASV12+mutations.">Kruppel-like factor 5 is a crucial mediator of intestinal tumorigenesis in mice harboring combined ApcMin and KRASV12 mutations.</a> Mol Cancer. 9 (63), (2010).</li><li>Pirici, D., 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=Antibody+elution+method+for+multiple+immunohistochemistry+on+primary+antibodies+raised+in+the+same+species+and+of+the+same+subtype.">Antibody elution method for multiple immunohistochemistry on primary antibodies raised in the same species and of the same subtype.</a> J Histochem Cytochem. 57 (6), 567-575 (2009).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> CSH Protoc. 2008, (2008).</li></ol>

The authors declare that they have no competing financial interests.

瑞士轧制技术是大规模准备组织学和形态学评估肠组织的有效方法。与此相反的先前描述瑞士轧制技术，其最初是为制备冷冻切片18,19的开发，这里介绍的过程允许福尔马林固定，石蜡包埋（FFPE）的提示肠组织制备和固定。相比冷冻组织，FFPE组织具有更长的保质期，是组织为由于更好组织的完整性的组织学分析的优选类型。瑞士轧协议的关键部分涉及滚动和维护瑞士卷的完整性和质量组?...

哺乳动物肠上皮包括柱状细胞的单层。在小肠中，增殖性细胞被局限于隐窝而分化细胞占据绒毛区域。然而，因为有在大肠没有绒毛，所述增殖性细胞被定位于隐窝的底部和分化的细胞占据隐窝的上部区域。肠上皮经历快速补货 - 由隐窝内的增殖细胞的连续分裂驱动（约3 5天）。隐窝的增殖细胞不是均匀的人口和进一步细分为干细胞和短暂扩充（TA）单元1。干细胞存在于隐窝的底部，前4内- 5细胞从最底部2。目前的模式支持两种类型的干细胞的存在:隐窝基底柱状（CBC）干细胞和储备静态干细胞。在CBC小号统的细胞活跃增殖和富含亮氨酸的含有重复序列的G蛋白偶联受体5（LGR5）3，Olfactomedin 4（OLFM4）4和Achaete盾状2（ASCL2）5被标记。另一方面，备用静态干细胞被B细胞特异性莫洛尼鼠白血病病毒整合位点1（Bmi1的）6，鼠标端粒酶逆转录酶（mTert）7标记，HOP同源框（Hopx）8，Doublecortin样和凸轮激酶样1（DCLK1）9，和亮氨酸-丰富的重复序列和免疫球蛋白样结构域1（LRIG1）10。在活跃增殖的干细胞产生TA细胞，然后进行进一步分化成吸收细胞（肠）和分泌细胞（肠内分泌，高脚杯，潘氏和簇绒细胞）。在增殖区连续细胞分裂导致沿着隐窝 - 绒毛轴上皮细胞的向上移动，直到它们到达绒毛，这里它们经受凋亡，并且是顶从上皮的表面脱落。不同类型的肠上皮细胞通过不同的蛋白质（ 例如 ，肠杯状细胞可通过用抗MUC2和帕内特细胞用抗溶菌酶抗体抗体染色识别）的表达标记。我们研究的Kruppel样因子（KLFs）在肠上皮11-13的稳态和病理学的作用。这里提出支撑改性瑞士滚动技术的可行性结果是基于Kruppel样因子的在维持活跃增殖肠上皮干细胞14的角色5（KLF5）的先前的研究。 KLF5是在活性肠干和TA细胞12高度表达的锌指转录因子。以前的研究表明，KLF5与Ki-67的，在肠隐窝已知增殖标记物共表达。 胃肠TR<html>行为不是结构上或功能上同质的组织。小肠分为十二指肠，空肠，回肠和大肠进入盲肠和结肠，后者进一步分成近侧，中间和远侧部分。每个部分都有独特的组织学特征，并播放不同的角色15。这样，损伤的效果和肠上皮细胞的反应程度可能取决于研究的组织16的区域。此外，各种小鼠品系表现出在基于在研究16中使用损伤的类型组织学水平的响应的多样性。从而，使之适合组织制剂是必要的，以允许适当的组织学和肠组织中的分子分析。因此，在同一时间的肠上皮细胞的完整长度的瑞士卷技术补助分析，从而ascertains基于综合信息灵通的结论。 e_content"&gt;瑞士辊技术首先由Magnus 17所提到的，并且由Moolenbeck和Ruitenberg和Park 等进行详细说明。作为制备组织和执行组织学方法中的啮齿动物肠18,19，分别协议的分析划定该出版物中提出了允许对用于诊断目的及时可靠的组织制备的原始方法的改进版本，这改性技术允许有效集合和制备肠上皮的对普遍使用的技术，如免疫组织化学，免疫荧光，以及原位杂交（荧光和发色20）。此外，经修饰的组织标本制备方法利用，同时提供快速组织固定的方法，容易获得的并且相对便宜的试剂，并允许蛋白质，DNA的恢复，和RNA为额外的评价。摘自同时，THI小号的技术是非常适合的肠上皮组织病理学，病理和分子特性的全面评估。

<table><tbody><tr><td>Stainless Steel Dissecting Kits</td><td>VWR</td><td>25640-002</td><td></td></tr><tr><td>Decloaking Chamber</td><td>Biocare Medical</td><td>DC2012</td><td></td></tr><tr><td>Syringe 10 ml</td><td>VWR</td><td>89215-218</td><td></td></tr><tr><td>Swingsette Tissue embedding/processing cassette with lid</td><td>Simport</td><td>M515</td><td></td></tr><tr><td>Superfrost Plus Slides [size: 25 x 75 x 1 mm]</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Manual Slide Staining Set</td><td>Tissue-Tek/Sakura</td><td>4451</td><td></td></tr><tr><td>Staining Dish Green</td><td>Tissue-Tek/Sakura</td><td>4456</td><td></td></tr><tr><td>Staining Dish White</td><td>Tissue-Tek/Sakura</td><td>4457</td><td></td></tr><tr><td>24-Slide Slide Holder with Detachable Handle</td><td>Tissue-Tek/Sakura</td><td>4465</td><td></td></tr><tr><td>Oven</td><td>Thermo Scientific</td><td>6243</td><td>for baking slides at 65 degree</td></tr><tr><td>Dissection microscope</td><td>Zeiss</td><td>Stemi 2000C</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>Eclipse 90i</td><td>Bright and fluoerescent light, with objectives: 10X, 20X</td></tr><tr><td>PAP Pen Super-Liquid Blocker Mini</td><td>Fisher Scientific</td><td>DAI-PAP-S-M</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>AAPR</td><td>111000200</td><td></td></tr><tr><td>Methanol</td><td>VWR</td><td>BDH1135-4LP</td><td></td></tr><tr><td>Glacial acetic acid</td><td>AAPR</td><td>281000ACS</td><td></td></tr><tr><td>Xylene</td><td>Fisher Scientific</td><td>X5P-1GAL</td><td></td></tr><tr><td>Hydrogen peroxide 25% solution in water</td><td>ACROS</td><td>202465000</td><td></td></tr><tr><td>10% bufered formalin</td><td>Fisher Scientific</td><td>22-026-213</td><td></td></tr><tr><td>Bovine serum fraction V, heat shock</td><td>Roche</td><td>3116956001</td><td></td></tr><tr><td>Tween 20</td><td>Sigma Aldrich</td><td>P7949</td><td></td></tr><tr><td>Sodium citrate</td><td>Fisher Scientific</td><td>S279</td><td></td></tr><tr><td>Gavage needle</td><td>VWR</td><td>20068-624</td><td></td></tr><tr><td>Rabbit anti Klf5 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-22797</td><td>Dilution 1:150</td></tr><tr><td>Chicken anti EGFP antibody</td><td>Millipore</td><td>AB16901</td><td>Dilution 1:500</td></tr><tr><td>Rabbit anti Ki67 antibody</td><td>Biocare Medical</td><td>CRM325B</td><td>Dilution 1:500</td></tr><tr><td>Mach3 rabbit AP polymer detection kit</td><td>Biocare Medical</td><td>M3R533L</td><td></td></tr><tr><td>Warp red chromogen kit</td><td>Biocare Medical</td><td>WR806 H</td><td></td></tr><tr><td>Lgr5-EGFP/CreER&lt;sup&gt;T2&lt;/sup&gt; mice&amp;nbsp;</td><td>Jackson labs</td><td>008875&amp;nbsp;</td><td></td></tr><tr><td>Automated processor</td><td>Leica</td><td>Leica TP1020</td><td></td></tr></tbody></table>

improved swiss-rolling technique for intestinal tissue preparation for immunohistochemical and immunofluorescent analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

在免疫组化染色结合瑞士轧制技术允许或大或小肠组织的全面分析。大肠一个C57BL / 6小鼠（图1）的H＆E染色的例子是可行性的图示和该技术的有效性。正如图1所示，该图像是能够捕捉到结肠的所有部分:近端，中间和远端。因此，它允许全面的组织学评估。瑞士轧制和捕捉小或大肠组织的整个长度的能力是异构的基因表达和标记染色或肠组织的侮?...

Watch this Scientific Journal Video about Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses at JoVE.com

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

准确识别，并沿着肠道粘膜内层的上皮细胞的位置是必要的，以定义不同的细胞谱系。肠组织的正常成像识别的蛋白表达模式与最大分辨率是至关重要的。本研究旨在描绘处理小鼠肠组织的最佳方法和条件。

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

improved-swiss-rolling-technique-for-intestinal-tissue-preparation

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

68.1K 次观看.  Stony Brook University School of Medicine. 准确识别，并沿着肠道粘膜内层的上皮细胞的位置是必要的，以定义不同的细胞谱系。肠组织的正常成像识别的蛋白表达模式与最大分辨率是至关重要的。本研究旨在描绘处理小鼠肠组织的最佳方法和条件。 

视频: Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Murine Colitis Modeling using Dextran Sulfate Sodium (DSS)

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of Crohn&rsquo;s Disease - Crohn&rsquo;s Colitis. Acute colitis may present with abdominal pain and distention, malabsorption, diarrhea, hematochezia and mucus in the stool. We are beginning to understand the complex interactions between the environment, genetics, and epithelial barrier dysfunction in Inflammatory Bowel Disease and animal models of colitis have been essential in advancing our understanding of this disease. One popular model involves supplementing the drinking water of mice with low-molecular weight Dextran Sodium Sulfate (DSS), resulting in epithelial damage and a robust inflammatory response in the colon lasting several days 1.Variations of this approach can be used to model acute injury, acute injury followed by repair, and repeated cycles of DSS interspersed with recovery modeling chronic inflammatory diseases 2. After a single four-day treatment of 3% DSS in drinking water, mice show signs of acute colitis including weight loss, bloody stools, and diarrhea. Mice are euthanized at the conclusion of the treatment course and at necropsy dissected colons are processed and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review.

Murine Colitis Modeling using Dextran Sulfate Sodium DSS

Dextran sulfate sodium (DSS) administered in the drinking water is an established murine inflammatory injury model of acute colitis. This protocol outlines the method for DSS treatment and the preparation of tissues.

小鼠结肠炎模型使用葡聚糖硫酸钠（DSS）

Colitis can occur from viral or bacterial infections, ischemic insult, or autoimmune disorders; most notably Ulcerative Colitis and the colonic variant of ...

科研

生物学

A Simple Device to Rapidly Prepare Whole Mounts of the Mouse Intestine

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe the use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations of superior and uniform quality to that which can be achieved by hand. The device comprises a base that holds 4 stainless steel rods and a top, which acts a cutting guide. The rods are inserted into the lumen of the small intestine [divided into thirds] and the colon. The rods and samples are then placed over a piece of filter paper or card into the holding slots in the base of the device. The top of the device is then positioned and serves as a cutting guide. The two angled sections in the center of the top piece are used to guide a knife or scalpel and cut the intestines longitudinally on the top of the rods. Once the intestinal sections have been cut, the top is removed and the card,&#160;tissue and rods gently removed from the device and placed on the bench. The rods are then gently rolled sideways to flatten and stick the intestinal segments onto the underlying piece of filter paper or card. The final preparation can then be examined or fixed and stored for later analysis. The preparations are invaluable for the study of intestinal changes in normal or genetically modified mouse models. The preparations have been used for the study and quantification of the effects of inflammation (colitis), damage, pre-cancerous lesions (aberrant crypt foci (ACFs) and mucin depleted foci (MDFs)) and polyps or tumors.

The use of a simple device to cut and &#8216;roll&#8217; mouse intestines to rapidly prepare whole mount preparations is described.

一个简单的设备，迅速准备小鼠肠的全坐骑

Preparing whole mounts of the mouse small intestine and colon for subsequent analysis or quantification can be time consuming and difficult. We describe ...

医学

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

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. However, robust standards for objective and reproducible quantification of disease severity remain to be defined. Most colitis analysis methods rely on limited histological scoring of small segments of intestine, leading to partial or biased analyses. Here, we combine high-resolution image acquisition and longitudinal analysis of the entire colon to quantify intestinal injury and ulceration in the dextran sodium sulfate (DSS) induced model of murine colitis. This protocol allows for the generation of objective and reproducible results without extensive user training. Here, we provide comprehensive details on sample preparation and image analysis using examples of data from DSS induced colitis. This method can be easily adapted to other models of murine colitis that have significant inflammation associated with mucosal injury. We demonstrate that the fraction of inflamed/injured and eroded/ulcerated mucosa relative to the complete length of the colon closely parallels clinical findings such as weight loss amid DSS-induced disease progression. This histological protocol provides a reliable time and cost-effective aid to standardize analyses of disease activity in an unbiased way in DSS colitis experiments.

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

Murine colitis models are tools that are extensively employed in studies focused on understanding the pathobiology of inflammatory intestinal disorders. ...

免疫与感染

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable health and financial burden on individuals and society. Therefore, it is critical to investigate the mechanisms underlying the pathogenesis and development of IBD. Here, a gut microbiota antigen-specific T cell transfer colitis model is described. CBir1 flagellin has been recognized as the immunodominant gut bacterial antigen in experimental colitis and patients with Crohn&#39;s disease. CBir1 TCR transgenic na&#970;ve CD4+ T cells, specific to CBir1 flagellin, can induce chronic colitis after adoptive transfer into immune-deficient Rag1-/- mice. The disease severity is assessed by histopathology. The CD4+ T cell phenotypes in colonic lamina propria are also determined. This model closely resembles the development of IBD, which provides an ideal murine model for investigating the mechanisms driving the pathogenesis of IBD and testing the potential drugs for treating IBD.

Induction of Intestinal Inflammation by Adoptive Transfer of CBir1 TCR Transgenic CD4+ T Cells to Immunodeficient Mice

In this protocol, a gut microbiota antigen-specific T cell adoptive transfer colitis model is described. CD4+ T cells are isolated from CBir1 TCR transgenic mice. These are specific for an immunodominant gut microbiota antigen CBir1 flagellin, which is transferred into recipient Rag1-/- mice, leading to intestinal inflammation.

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

With the increase of incidence, inflammatory bowel diseases (IBD), which are chronic diseases affecting the gastrointestinal tract, impose a considerable ...

Use of Anti-phospho-girdin Antibodies to Visualize Intestinal Tuft Cells in Free-Floating Mouse Jejunum Cryosections

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is phosphorylated by both receptor and non-receptor tyrosine kinases at tyrosine 1798. Omori et al. developed site- and phosphorylation status-specific antibodies against human girdin at tyrosine-1798 (pY1798), which specifically bind to phosphorylated tyrosine-1798, but not to unphosphorylated tyrosine-1798. pY1798 antibodies have been used to specifically label tuft cells (TCs) that are present in mammalian gastrointestinal tissues, but the function of these cells is unclear. This protocol allows the robust visualization of TCs in the jejunum using pY1798 antibodies and immunofluorescence. To ensure successful and simple TC visualization, this protocol includes two histological techniques: production of free-floating cryosections from gelatin-filled jejunum tissue, and low-temperature antigen retrieval at 50 &#176;C for 3 h. Filling the jejunum with gelatin maintains the shape of free-floating sections throughout the staining procedure, whereas low-temperature antigen retrieval ensures robust signals from TCs. Successful use of this protocol results in pY1798 staining of TCs distributed from villus tip to crypt. Stained TCs have a spool-shaped soma and fluorescent signals condense at the lumenal tip, which corresponds to the protruding &#39;tuft.&#39;&#160;Phalloidin staining colocalized with pY1798-positive TCs at the thickened brush border, and corresponds to a rootlet mass extending from the TC tuft. This protocol could be used to examine TCs in human biopsy samples collected with gastrointestinal endoscopes. Furthermore, TCs were recently reported to accumulate following parasite infection in mice, suggesting that this protocol could have applications for diagnosis of parasite infections in the human gut.

Kuga et al. discovered that phosphorylation-status specific antibodies against the actin binding protein girdin phosphorylated at tyrosine 1798 (pY1798) can be used to label tuft cells (TCs). This protocol allows robust visualization of TCs using immunofluorescent staining of free-floating jejunum cryosections with pY1798 antibodies.

抗磷 girdin 抗体在自由漂浮小鼠空肠 Cryosections 小肠簇细胞中的应用

The actin binding protein girdin is a cytosolic protein that is required for actin remodeling to trigger cell migration in various tissues. Girdin is ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

用于肠道组织高质量免疫染色的高效 Swiss Roll 方法

Optimized Protocol for Intestinal Swiss Rolls and Immunofluorescent Staining of Paraffin Embedded Tissue

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ileum. Each anatomical region of the intestine has a unique function that is reflected by differences in cellular structure. Investigating changes in the intestine requires an in-depth analysis of different tissue regions and cellular alterations. To study the intestine and visualize large pieces of tissue, researchers commonly use a technique known as intestinal Swiss rolls. In this technique, the intestine is divided into each anatomical region and fixed in a flat orientation. Then, the tissue is carefully rolled and processed for paraffin embedding. Proper tissue fixation and orientation is an often-overlooked laboratory technique but is critically important for downstream analysis. Additionally, improper Swiss rolling of intestinal tissue can damage the fragile intestinal epithelium, leading to poor tissue quality for immunostaining. Ensuring well-fixed and properly oriented tissue with intact cellular structures is a crucial step that ensures optimal visualization of intestinal cells. We present a cost-effective and simple method for making Swiss rolls to include all sections of the intestine in a single paraffin-embedded block. We also describe optimized immunofluorescence staining of intestinal tissue to study various aspects of the intestinal epithelium. The following protocol provides researchers with a comprehensive guide to obtaining high-quality immunofluorescence images through intestinal tissue fixation, Swiss-roll technique, and immunostaining. Employing these refined approaches preserves the intricate morphology of the intestinal epithelium and fosters a deeper understanding of intestinal physiology and pathobiology.

作者聚焦：用于肠道细胞生物学研究的有效免疫荧光染色方案

The intestine is vital for digestion and absorption. Each region-duodenum, jejunum, ileum, colon-serves distinct functions due to unique cellular structures. Studying intestinal physiology demands meticulous tissue analysis. This protocol outlines tissue fixation and processing using the Swiss roll technique, ensuring accurate immunostaining through proper tissue preservation and orientation.

用于肠瑞士卷和石蜡包埋组织的免疫荧光染色的优化方案

The intestine is a complex organ composed of the small and the large intestines. The small intestine can be further divided into duodenum, jejunum, and ...

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

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小鼠结肠炎模型使用葡聚糖硫酸钠（DSS）

一个简单的设备，迅速准备小鼠肠的全坐骑

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

通过将CBir1 TCR转基因CD4 ⁺ T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

抗磷 girdin 抗体在自由漂浮小鼠空肠 Cryosections 小肠簇细胞中的应用

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

用于肠瑞士卷和石蜡包埋组织的免疫荧光染色的优化方案

用于肠瑞士卷和石蜡包埋组织的免疫荧光染色的优化方案

瑞士改进轧制技术肠组织准备免疫组化和免疫分析

摘要

探索更多视频

此视频中的章节

小鼠结肠炎模型使用葡聚糖硫酸钠（DSS）

一个简单的设备，迅速准备小鼠肠的全坐骑

激光捕获显微切割分离新鲜冷冻人体肠神经节的优化

穆林·德克斯特兰硫酸钠诱导结肠炎模型中肠道炎症的系统评分分析

通过荧光 原位 杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

通过将CBir1 TCR转基因CD4 + T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症

抗磷 girdin 抗体在自由漂浮小鼠空肠 Cryosections 小肠簇细胞中的应用

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

用于肠瑞士卷和石蜡包埋组织的免疫荧光染色的优化方案

用于肠瑞士卷和石蜡包埋组织的免疫荧光染色的优化方案

通过荧光原位杂交、凝集素染色和成像可视化肠道微生物群与宿主的相互作用

通过将CBir1 TCR转基因CD4 ⁺ T细胞过继转移到免疫缺陷小鼠来诱导肠道炎症