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>

<ol>
	<li>van der Flier, L. G., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells,+self-renewal,+and+differentiation+in+the+intestinal+epithelium.">Stem cells, self-renewal, and differentiation in the intestinal epithelium.</a> Annu Rev Physiol. 71, 241-260 (2009).</li><li>Bjerknes, M., Cheng, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+isolation+of+intact+epithelium+from+the+mouse+intestine.">Methods for the isolation of intact epithelium from the mouse intestine.</a> Anat Rec. 199, 565-574 (1981).</li><li>Barker, 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=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449 (7165), 1003-1007 (2007).</li><li>van der Flier, L. G., Haegebarth, A., Stange, D. E., van de Wetering, M., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OLFM4+is+a+robust+marker+for+stem+cells+in+human+intestine+and+marks+a+subset+of+colorectal+cancer+cells.">OLFM4 is a robust marker for stem cells in human intestine and marks a subset of colorectal cancer cells.</a> Gastroenterology. 137 (1), 15-17 (2009).</li><li>van der Flier, L. G., 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=Transcription+factor+achaete+scute-like+2+controls+intestinal+stem+cell+fate.">Transcription factor achaete scute-like 2 controls intestinal stem cell fate.</a> Cell. 136 (5), 903-912 (2009).</li><li>Sangiorgi, E., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bmi1+is+expressed+in+vivo+in+intestinal+stem+cells.">Bmi1 is expressed in vivo in intestinal stem cells.</a> Nat Genet. 40 (7), 915-920 (2008).</li><li>Montgomery, R. K., 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=Mouse+telomerase+reverse+transcriptase+(mTert)+expression+marks+slowly+cycling+intestinal+stem+cells.">Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells.</a> Proc Natl Acad Sci U S A. 108 (1), 179-184 (2011).</li><li>Takeda, 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=Interconversion+between+intestinal+stem+cell+populations+in+distinct+niches.">Interconversion between intestinal stem cell populations in distinct niches.</a> Science. 334 (6061), 1420-1424 (2011).</li><li>May, 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=Identification+of+a+novel+putative+gastrointestinal+stem+cell+and+adenoma+stem+cell+marker,+doublecortin+and+CaM+kinase-like-1,+following+radiation+injury+and+in+adenomatous+polyposis+coli/multiple+intestinal+neoplasia+mice.">Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice.</a> Stem Cells. 26 (3), 630-637 (2008).</li><li>Powell, A. E., 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+pan-ErbB+negative+regulator+Lrig1+is+an+intestinal+stem+cell+marker+that+functions+as+a+tumor+suppressor.">The pan-ErbB negative regulator Lrig1 is an intestinal stem cell marker that functions as a tumor suppressor.</a> Cell. 149 (1), 146-158 (2012).</li><li>Pearson, R., Fleetwood, J., Eaton, S., Crossley, M., Bao, S. <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+transcription+factors:+a+functional+family.">Kruppel-like transcription factors: a functional family.</a> Int J Biochem Cell Biol. 40 (10), 1996-2001 (2008).</li><li>McConnell, B. B., Yang, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+Kruppel-like+factors+in+health+and+diseases.">Mammalian Kruppel-like factors in health and diseases.</a> Physiol Rev. 90 (4), 1337-1381 (2010).</li><li>McConnell, B. B., Ghaleb, A. M., Nandan, M. O., Yang, V. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diverse+functions+of+Kruppel-like+factors+4+and+5+in+epithelial+biology+and+pathobiology.">The diverse functions of Kruppel-like factors 4 and 5 in epithelial biology and pathobiology.</a> Bioessays. 29 (6), 549-557 (2007).</li><li>Nandan, M. O., Ghaleb, A. M., Bialkowska, A. B., Yang, V. W. <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+essential+for+proliferation+and+survival+of+mouse+intestinal+epithelial+stem+cells.">Kruppel-like factor 5 is essential for proliferation and survival of mouse intestinal epithelial stem cells.</a> Stem Cell Res. 14 (1), 10-19 (2015).</li><li>Gelberg, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+anatomy,+physiology,+and+mechanisms+of+disease+production+of+the+esophagus,+stomach,+and+small+intestine.">Comparative anatomy, physiology, and mechanisms of disease production of the esophagus, stomach, and small intestine.</a> Toxicol Pathol. 42 (1), 54-66 (2014).</li><li>De Robertis, M., 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+AOM/DSS+murine+model+for+the+study+of+colon+carcinogenesis:+From+pathways+to+diagnosis+and+therapy+studies.">The AOM/DSS murine model for the study of colon carcinogenesis: From pathways to diagnosis and therapy studies.</a> J Carcinog. 10 (9), (2011).</li><li>Magnus, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Observations+on+the+presence+of+intestinal+epithelium+in+the+gastric+mucosa.">Observations on the presence of intestinal epithelium in the gastric mucosa.</a> The Journal of Pathology and Bacteriology. 44 (2), 389-398 (1937).</li><li>Moolenbeek, C., Ruitenberg, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+"Swiss+roll":+a+simple+technique+for+histological+studies+of+the+rodent+intestine.">The "Swiss roll": a simple technique for histological studies of the rodent intestine.</a> Lab Anim. 15 (1), 57-59 (1981).</li><li>Park, C. M., Reid, P. E., Walker, D. C., MacPherson, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+practical+'swiss+roll'+method+of+preparing+tissues+for+paraffin+or+methacrylate+embedding.">A simple, practical 'swiss roll' method of preparing tissues for paraffin or methacrylate embedding.</a> J Microsc. 145, 115-120 (1987).</li><li>Summersgill, B., Clark, J., Shipley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+and+chromogenic+in+situ+hybridization+to+detect+genetic+aberrations+in+formalin-fixed+paraffin+embedded+material,+including+tissue+microarrays.">Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays.</a> Nat Protoc. 3 (2), 220-234 (2008).</li><li>Takeda, 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=Cardiac+fibroblasts+are+essential+for+the+adaptive+response+of+the+murine+heart+to+pressure+overload.">Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload.</a> J Clin Invest. 120 (1), 254-265 (2010).</li><li>el Marjou, F., 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=Tissue-specific+and+inducible+Cre-mediated+recombination+in+the+gut+epithelium.">Tissue-specific and inducible Cre-mediated recombination in the gut epithelium.</a> Genesis. 39 (3), 186-193 (2004).</li><li>McConnell, B. 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织染色postfixation组织的灵活性。对于肠组织的FFPE标准瑞士辊技术通常是费力和耗时（2天）18,19。此外，该标准方法通常会产生肠组织中为relatively僵硬，不容易形成在瑞士卷。这是因为组织轧制之前需要在10％缓冲福尔马林解剖肠组织的保温过夜。对FFPE目的的技术的其它修改也已被开发，但它们有肠组织的倾向的一个主要问题，以展开和/或用于辊的中心被扭曲。这是因为，在这些技术中使用的组织为新鲜未定影组织，这是从由肠产生的粘液滑。这里示出的新改进的方法，通过使用布安固定液的改进形式克服了这些问题。原始布安固定液包括乙酸，乙醇，苦味酸和多聚甲醛（或甲醛）的混合物。两个最有害成分，苦味酸和多聚甲醛（或甲醛），已被淘汰，以允许与乙酸/乙醇混合更安全使用的固定剂。苦味酸呈现爆炸的危险，并且paraformaldehyde（或福尔马林）是癌症的危险。使用修改的Bouin的固定液的意义在于，它（1）是危险性较小相比，它的原配方，（2）很容易与相对nonexpensive试剂是在大多数实验室容易获得的准备，和（3）允许快速，几乎是一瞬间，用于冲洗当组织的固定。知识，还没有已知的限制，使用该修改的技术。此外，快速定影这种混合物极大地减少了肠组织的滑溜，从而更快，更容易组织碾压。这固定液还允许组织和细胞的完整性保护优秀组织学和免疫分析。因此，相较于肠组织制备的其它方法，该改进的技术克服了几个技术问题，并授予大大提高速度和易用性。 此外，这种改性固定液是有用与其它厚使用需要快速定影组织。由于乙醇乙酸和性质，具有厚的组织的快速渗透的能力，而在同一时间进行的固定。我们已经成功地用它来快速修复厚组织，如肝脏，脾脏和肾脏。 20分钟 - 例如，使用改性布安固定液成年小鼠整个肝脏的固定器15内通常实现。这可以通过切割的截面进入肝脏和观察肝脏的深区域是否已经改变从血红色到灰色容易地判断。颜色的变化表明固定。然后，这个固定其次是用福尔马林24交联 - 无惧改变细胞/组织构成的48小时，作为组织已经固定在了这一点。 相比之下，使用传统的缓冲，福尔马林固定的成年小鼠全肝单独方法将需要24 - 48小时达到深层组织fixati上，与深部组织的改变的细胞的组合物的风险。厚组织的快速固定方法具有减少到最低的细胞成分的改变响应于应力条件下，如缺氧，在除去从身体的器官/组织的优越优点。我们预计，改性固定液可在动物灌注用作期望定影厚的组织/器官的更快的方法。因此，在结束时，该方法和修饰的固定剂在用于肠组织收获和固定和改性固定液传统的方法提供了多个优点，可用于快速固定其他厚组织/器官。

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 border="0" cellpadding="0" cellspacing="0" width="1016">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Stainless Steel Dissecting Kits</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">25640-002</td>
			<td style="width:403px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Decloaking Chamber</td>
			<td style="width:179px;">Biocare Medical</td>
			<td>DC2012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Syringe 10ml</td>
			<td style="width:179px;">VWR</td>
			<td>89215-218</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:316px;">Swingsette Tissue embedding/processing cassette with lid</td>
			<td style="width:179px;">Simport</td>
			<td>M515</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Superfrost Plus Slides [size: 25x75x1mm]</td>
			<td style="width:179px;">VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Manual Slide Staining Set</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Staining Dish Green</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4456</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Staining Dish White</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4457</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">24-Slide Slide Holder with Detachable Handle</td>
			<td style="width:179px;">Tissue-Tek/Sakura</td>
			<td>4465</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Oven</td>
			<td style="width:179px;">Thermo Scientific</td>
			<td>6243</td>
			<td>for baking slides at 65 degree</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Dissection microscope</td>
			<td style="width:179px;">Zeiss</td>
			<td>Stemi 2000C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Microscope</td>
			<td style="width:179px;">Nikon</td>
			<td>Eclipse 90i</td>
			<td>Bright and fluoerescent light, with objectives: 10x, 20x</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">PAP Pen Super-Liquid Blocker Mini</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>DAI-PAP-S-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Ethanol 200 proof</td>
			<td style="width:179px;">AAPR</td>
			<td>111000200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Methanol</td>
			<td style="width:179px;">VWR</td>
			<td>BDH1135-4LP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Glacial acetic acid</td>
			<td style="width:179px;">AAPR</td>
			<td>281000ACS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Xylene</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>X5P-1GAL</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Hydrogen peroxide 25% solution in water</td>
			<td style="width:179px;">ACROS</td>
			<td>202465000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">10% bufered formalin</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td>22-026-213</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Bovine serum fraction V, heat shock</td>
			<td style="width:179px;">Roche</td>
			<td>3116956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Tween 20</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td>P7949</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Sodium citrate</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">S279</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Gavage needle</td>
			<td style="width:179px;">VWR</td>
			<td style="width:119px;">20068-624</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rabbit anti Klf5 antibody</td>
			<td style="width:179px;">Santa Cruz Biotechnology</td>
			<td style="width:119px;">sc-22797</td>
			<td>Dilution 1: 150</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Chicken anti EGFP antibody</td>
			<td style="width:179px;">Millipore</td>
			<td style="width:119px;">AB16901</td>
			<td>Dilution 1: 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Rabbit anti Ki67 antibody</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">CRM325B</td>
			<td>Dilution 1: 500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Mach3 rabbit AP polymer detection kit</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">M3R533L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Warp red chromogen kit</td>
			<td style="width:179px;">Biocare Medical</td>
			<td style="width:119px;">WR806 H</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Lgr5-EGFP/CreERT2 mice&#160;</td>
			<td>Jackson labs</td>
			<td>008875&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Automated processor</td>
			<td style="width:179px;">Leica</td>
			<td style="width:119px;">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辱可变响应非常有帮助。 一个例子是在LGR5-EGFP / CreER T2小鼠EGFP染色模式。在该小鼠模型中，EGFP表达被驱动LGR5启动子，其是活性仅在活跃增殖肠隐窝。此外，此小鼠模型的特点是低 - 转基因表达的（约5 10％）外显率。正如图2所示，在肠道组织中的EGFP表达模式是可变的。因此，为了捕获所述组织的一个大视图的能力有助于确定感兴趣的区域。 此处所描述的技术是强有力的特别是与multifluorophore染色的应用。这里，我们表明，用的是瑞士的辊技术制备的肠组织的trifluorophore染色的一个实例。本研究的主要目的是调查KLF5在维持表达LGR5标记肠干细胞的作用。因此，我们从LGR5-EGFP / CreER T2小鼠LGR5阳性肠道干细胞删除KLF5和收集一天肠组织14第一次他莫昔芬注射后。对于免疫组织学分析，将组织按照本手稿提出的协议，和染色的EGFP（LGR5标记物），KLF5，和Ki-67进行制备。在对照小鼠，记为LGR5-EGFP / CreER T2，两者的EGFP阳性（标有蓝色箭头）和EGFP阴性隐窝（标有白箭头）显示出两个检测时间点共染色为KLF5和Ki-67 ， 如图3D所示。与此相反，在LGR5-EGFP / CreER T2 / KLF5 FL / FL小肠组织，KLF5 / Ki-67的共染色从EGFP阳性CBC干细胞缺失（由蓝色箭头标记），但存在于EGFP阴性隐窝邻接绿色隐窝（白色箭头标记）， 如图3H。这是一个很好的例子，这里介绍的组织制备技术不产生负面影响染色质量。 从 C57B L / 6鼠标，显示一张大肠 图1. H＆E染色是大肠的整个长度的合成图像。黑色箭头和黑线之间的部分标记近端部分之间的黑色和橙色线痕的中间，和橙色线和橙色箭头之间标志着大肠的末端部分。比例尺= 1000微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54161/54161fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54161/54161fig2.jpg" /> 图2. LGR5-EGFP / CreER T2 鼠标小肠的免疫荧光染色 的 E复合图像的LGR5-EGFP / CreER T2小鼠的小肠切片的GFP（标记LGR5阳性上皮细胞）染色。该标签LGR5-阳性上皮细胞在LGR5-EGFP / CreER T2鼠标隐窝蛋白标记（EGFP）的异构免疫荧光染色的例子。细胞核可视化与赫斯特。白色箭头标记隐窝阳性表达EGFP。比例尺= 500微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54161/54161fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54161/54161fig3.jpg" /> 图 3. 删除 KLF5 在LGR5-EGFP / CreER T2 / KLF5 FL / FL 小鼠坚持LGR5-EGFP阳性囊长远。 </st荣&gt;顶部和底部图像组是从LGR5-EGFP / CreER T2控制和LGR5-EGFP / CreER T2 / KLF5 FL / FL上天的小鼠分别为14天第一次他莫昔芬注射后，代表小肠组织。面板AD代表性染色从LGR5-EGFP / CreER T2对照组小鼠采集的组织，而代表LGR5-EGFP / CreER T2的面板EH显示染色/ KLF5 FL / FL小鼠。图A和E显示KLF5免疫荧光染色为红色;面板B和F示出了绿色EGFP染色;在黄色面板C和G显示Ki-67的染色;和面板D和H显示合并KLF5，EGFP和Ki-67的污渍图像。蓝色箭头指向绿色隐窝;白色箭头指向合并后的图像非绿色隐窝。 LGR5-EGFP / CreER T2 / KLF5 FL /˚F升小鼠显示出仅在EGFP标记的CBC细胞14 KLF5的长期损失。比例尺= 50微米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54161/54161fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

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

Nanyang Technological University

Research

JoVE Journal

Biology

68.0K 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

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

改进的膜片钳记录的压力抛光移液器

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

科研

生物学

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

小学组织两个胸苷类似物的免疫荧光检测（CldU IDU）

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

Evisceration of Mouse Vitreous and Retina for Proteomic Analyses

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is a highly aqueous extracellular matrix overlying the retina where intraocular as well as extraocular proteins accumulate during disease.1-3 Abnormal interactions between vitreous and retina underlie several diseases such as retinal detachment, proliferative diabetic retinopathy, uveitis, and proliferative vitreoretinopathy.1,4 The relative mouse vitreous volume is significantly smaller than the human vitreous (Figure 1), since the mouse lens occupies nearly 75% of its eye.5 This has made biochemical studies of mouse vitreous challenging. In this video article, we present a technique to dissect and isolate the mouse vitreous from the retina, which will allow use of transgenic mouse models to more clearly define the role of this extracellular matrix in the development of vitreoretinal diseases.

The dissection technique illustrates evisceration of the vitreous, retina, and lens from the mouse eye, separation by centrifugation, and characterization with protein assays.

眼内容剜除小鼠玻璃体和视网膜蛋白质组分析

While the mouse retina has emerged as an important genetic model for inherited retinal disease, the mouse vitreous remains to be explored. The vitreous is ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

简单和高效的睾丸细胞悬液的制备技术

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

轨迹数据分析行人时空活性研究

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

An Immunofluorescent Method for Characterization of Barrett&#8217;s Esophagus Cells

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One of the primary risk factors of EAC is Barrett&#8217;s esophagus (BE), a metaplastic change of the normal squamous esophagus in response to chronic heartburn. Despite the well-established connection between EAC and BE, interrogation of the molecular events, particularly altered signaling pathways involving progression of BE to EAC, are poorly understood. Much of this is due to the lack of suitable in vitro models available to study these diseases. Recently, immortalized BE cell lines have become commercially available allowing for in vitro studies of BE. Here, we present a method for immunofluorescent staining of immortalized BE cell lines, allowing in vitro characterization of cell signaling and structure after exposure to therapeutic compounds. Application of these techniques will help develop insight into the mechanisms involved in BE to EAC progression and provide potential avenues for treatment and prevention of EAC.

There is a discernible need for improved information on molecular drivers of Barrett&#8217;s Esophagus. Immunofluorescent staining is a useful technique for understanding the effects of cell signaling on cell morphology. We present a simple, effective protocol for the use of immunofluorescent staining to assess therapeutic treatment in Barrett&#8217;s Esophagus cells.

免疫荧光法Barrett食管细胞的表征

Esophageal adenocarcinoma (EAC) has an overall survival rate of less than 17% and incidence of EAC has risen dramatically over the past two decades. One ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

混合式切割:用于顽拗植物组织样品的改进切法

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

气孔带皮: 一种改进的防护细胞样品制备方法

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...