使用免疫荧光对患有TNBS诱导克罗恩病的小鼠结肠雌激素受体的可视化
1Department of Cytobiochemistry, Faculty of Biology and Environmental Protection,University of Lodz, 2Department of Biochemistry, Faculty of Medicine,Medical University of Lodz, 3Laboratory of Microscopic Imaging and Specialized Biological Techniques, Faculty of Biology and Environmental Protection,University of Lodz
Summary
该协议提出了完整的经验证的克罗恩病TNBS诱导的鼠模型，以及利用嵌入在石蜡中的正式固定结肠部分的免疫荧光，通过免疫组织化学对雌激素受体进行可视化的方法。
Abstract
克罗恩病是诊断最多的炎症性肠病。肠道中发育的慢性炎症导致蠕动紊乱和肠道粘膜损伤，似乎与结肠肿瘤转化风险增加有关。累积证据表明，雌激素和雌激素受体不仅影响对激素敏感的组织，还影响与雌激素没有直接关系的其他组织，如肺或结肠。在这里，我们描述了从TNBS诱发的克罗恩病的鼠模型中获得结肠雌激素受体成功免疫荧光染色的规程。提供了小鼠和肠道制剂中克罗恩病诱导的详细方案，以及使用正式固定石蜡内嵌肠部分的逐步免疫组织化学程序。所述方法不仅可用于体内雌激素受体检测和雌激素信号研究，还可用于可能参与结肠炎发展的其他蛋白质。
Introduction
克罗恩病 （CD） 是一种炎症性肠病 （IBD），表现为慢性肠道炎症。CD的病因知之甚少，但似乎有几个主要因素导致CD发育，包括肠道微生物群，以及遗传和环境因素，如饮食或压力1。为了更好地理解克罗恩病的发病机制，使用了几种肠道炎症模型，2、3、4、5、6、7。2,3,4,5,6,7在本文中，我们介绍了从2，4，6-三硝基苯磺酸（TNBS）诱导的CD的鼠模型中获得的结果。
据记载，雌激素能够调节慢性肠道炎症898，9，10，11，12。10,11,12,雌激素的生物活性由同源受体介导，其中包括核雌激素受体（ERs），即ER®（基因ESR1）和ER®（基因ESR2），以及G蛋白耦合雌激素受体，即G蛋白耦合雌激素受体，即GPER（基因GPER1），称为膜结合ER13，14。,14有几种方法可以确定雌激素受体的水平，但只有一些可用于在肠道中可视化它们。
免疫组织化学（IHC）是一种广泛用于临床和基础研究的方法，用于检测具有氟铬偶联抗体的细胞或组织中的某些抗原。IHC似乎是组织结构可视化以及特定蛋白质的识别和定位的重要方法，对于了解结肠炎的发展可能至关重要。在这里，我们提出了一个完整的和验证的方案，用于免疫荧光对肠道中雌激素受体的免疫组织化学可视化。
Protocol
动物研究是根据欧洲议会和理事会2010年9月22日第2010/63/欧盟指令以及机构建议，在地方伦理委员会（28/[B29/2016）》同意下进行的。 1. 克罗恩病的TNBS诱导的鼠型 注：本协议使用雄性BALB/C小鼠体重25-28克。动物被安置在恒定温度（22-24 °C）和相对湿度55×5%，并保持在12小时光/暗周期，免费获得标准粒状物和自来水。 将鼠标放入感应室并紧紧闭?…
Representative Results
TNBS诱发克罗恩病小鼠结肠的宏观特征图2显示了从对照和TNBS处理小鼠身上拍摄的结肠的代表性图像。在具有TNBS诱导的克罗恩病模型的小鼠中，结肠的长度减少，而结肠的宽度增加。 图2：从对照小鼠（对照）和TNBS治疗?…
Discussion
IBD病理生理学检查有许多动物模型，包括遗传、免疫或自发模型，以及化学诱导模型15。在几种类型动物模型中，如本协议中描述的TNBS诱导模型等化学诱导模型相对便宜且易于获得。TNBS诱导的结肠炎的鼠型具有与CD病理基础相关的几种临床症状。带诱导性结肠炎的动物的特点是粪便形成不一致，腹泻和体重减轻。然而，这并不意味着该模型可用于专门研究CD病因。TNBS诱导模型被?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
由于洛兹大学当局的财政支助，这项工作发表得有：科学研究副校长、国家和国际合作副校长和生物和环境保护学院院长。达米安·杰塞尼克得到波兰国家科学中心的赠款（2017/24/T/NZ5/00045和2015/17/N/NZ5/00336）的支持。
Materials
| Animals | |||
| BALB/C mice | University of Lodz | NA | |
| Equipment | |||
| Caliper | VWR | 62379-531 | |
| Cardboard block | NA | NA | |
| Confocal microscope – TCS SP8 | Leica Biosystems | NA | |
| Fully automated rotary microtome – RM2255 | Leica Biosystems | NA | |
| Glass slide | Thermo Scientific | J1800BMNT | |
| Heated Paraffin Embedding Module – EG1150 H | Leica Biosystems | NA | |
| Histological box | Marfour | LN.138747 | |
| Hydrophobic pen | Sigma-Aldrich | Z377821 | |
| Laboratory balance | Radwag | WL-104-0048 | |
| LAS X software | Leica Biosystems | NA | |
| Metal mold | Marfour | CP.5105 | |
| Sterile gauze | NA | NA | |
| Sterile scissor | NA | NA | |
| Sterile tweezer | NA | NA | |
| Tissue processor – TP1020 | Leica Biosystems | NA | |
| Reagents | |||
| 2, 4, 6-trinitrobenzene sulfonic acid | Sigma-Aldrich | 92822 | |
| Bovine serum albumin | Sigma-Aldrich | A3294 | |
| DiOC6 (3) | Sigma-Aldrich | 318426 | |
| DyLight 650 secondary antibody | Abcam | ab96886 | |
| ERα primary antibody | Abcam | ab75635 | |
| ERβ primary antibody | Abcam | ab3576 | |
| Ethanol | Avantor Performance Materials Poland | 396480111 | |
| Formaldehyde | Avantor Performance Materials Poland | 432173111 | |
| GPER primary antibody | Abcam | ab39742 | |
| Hydrochloric acid | Avantor Performance Materials Poland | 575283421 | |
| Hydrogen peroxidase | Avantor Performance Materials Poland | 885193111 | |
| isoflurane (forane) | Baxter | 1001936040 | |
| Normal goat serum | Gibco | 16210064 | |
| Paraffin | Leica Biosystems | 39602012 | |
| Petrie dish | Nest Scientific | 705001 | |
| Phosphate buffer saline | Sigma-Aldrich | P3813 | |
| Physiological saline | Sigma-Aldrich | 7982 | |
| Primocin (antibiotic) | Invitrogen | ant-pm-1 | |
| ProLong Diamond Antifage Mountant with DAPI (glycerol-based liquid with DAPI) | Invitrogen | P36971 | |
| Sodium chloride | Chempur | WE/231-598-3 | |
| Sodium citrate | Avantor Performance Materials Poland | 795780429 | |
| Tris | Avantor Performance Materials Poland | 853470115 | |
| Triton X-100 | Sigma-Aldrich | T8787 | |
| Tween 20 | Sigma-Aldrich | P9416 | |
| Xylene | Avantor Performance Materials Poland | BA0860119 |
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Citer Cet Article
Jacenik, D., Zielińska, M., Michlewska, S., Fichna, J., Krajewska, W. M. Visualization of Estrogen Receptors in Colons of Mice with TNBS-Induced Crohn’s Disease using Immunofluorescence. J. Vis. Exp. (157), e60813, doi:10.3791/60813 (2020).