瑞士改进轧制技术肠组织准备免疫组化和免疫分析
Summary
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。
Abstract
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.
Introduction
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“>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。Protocol
Representative Results
Discussion
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?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to thank Ainara Ruiz de Sabando for providing H&E images. This work was supported by grants from the National Institutes of Health (DK052230, DK093680 and CA172113) awarded to Dr. Vincent W. Yang.
Materials
| Stainless Steel Dissecting Kits | VWR | 25640-002 | |
| Decloaking Chamber | Biocare Medical | DC2012 | |
| Syringe 10ml | VWR | 89215-218 | |
| Swingsette Tissue embedding/processing cassette with lid | Simport | M515 | |
| Superfrost Plus Slides [size: 25x75x1mm] | VWR | 48311-703 | |
| Manual Slide Staining Set | Tissue-Tek/Sakura | 4451 | |
| Staining Dish Green | Tissue-Tek/Sakura | 4456 | |
| Staining Dish White | Tissue-Tek/Sakura | 4457 | |
| 24-Slide Slide Holder with Detachable Handle | Tissue-Tek/Sakura | 4465 | |
| Oven | Thermo Scientific | 6243 | for baking slides at 65 degree |
| Dissection microscope | Zeiss | Stemi 2000C | |
| Fluorescence Microscope | Nikon | Eclipse 90i | Bright and fluoerescent light, with objectives: 10x, 20x |
| PAP Pen Super-Liquid Blocker Mini | Fisher Scientific | DAI-PAP-S-M | |
| Ethanol 200 proof | AAPR | 111000200 | |
| Methanol | VWR | BDH1135-4LP | |
| Glacial acetic acid | AAPR | 281000ACS | |
| Xylene | Fisher Scientific | X5P-1GAL | |
| Hydrogen peroxide 25% solution in water | ACROS | 202465000 | |
| 10% bufered formalin | Fisher Scientific | 22-026-213 | |
| Bovine serum fraction V, heat shock | Roche | 3116956001 | |
| Tween 20 | Sigma Aldrich | P7949 | |
| Sodium citrate | Fisher Scientific | S279 | |
| Gavage needle | VWR | 20068-624 | |
| Rabbit anti Klf5 antibody | Santa Cruz Biotechnology | sc-22797 | Dilution 1: 150 |
| Chicken anti EGFP antibody | Millipore | AB16901 | Dilution 1: 500 |
| Rabbit anti Ki67 antibody | Biocare Medical | CRM325B | Dilution 1: 500 |
| Mach3 rabbit AP polymer detection kit | Biocare Medical | M3R533L | |
| Warp red chromogen kit | Biocare Medical | WR806 H | |
| Lgr5-EGFP/CreERT2 mice | Jackson labs | 008875 | |
| Automated processor | Leica | Leica TP1020 |
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