通过多色免疫测定法对过敏性鼻炎大鼠鼻粘膜组织切片进行免疫荧光标记
Summary
该方案描述了一种用于评估过敏性鼻炎大鼠模型的多色免疫荧光技术。
Abstract
过敏性鼻炎 （AR） 是一种慢性、非感染性鼻粘膜炎症性疾病，主要由特异性免疫球蛋白 E （IgE） 介导，影响全球约 10%-20% 的人口。虽然免疫荧光 （IF） 染色长期以来一直是检测疾病特异性蛋白质表达的标准技术，但传统的 IF 技术在检测同一样品中三种或更多蛋白质的表达水平的能力方面受到限制。因此，近年来开发了多色IF技术，可以同时标记细胞或组织中的多个靶标。
该协议全面概述了建立AR大鼠模型，获取鼻粘膜样本以及多色免疫荧光技术程序的过程。AR组所有大鼠均出现打喷嚏、流鼻涕、鼻痒等典型症状，行为观察得分为≥5分。苏木精和伊红 （H&E） 染色显示 AR 组的炎症细胞计数增加并破坏鼻粘膜完整性。多色免疫荧光（mIF）显示AR大鼠鼻黏膜组织中RORγt和TICAM-1的表达增加，而Foxp3的表达降低。
Introduction
过敏性鼻炎 （AR） 是一种慢性、非感染性鼻粘膜炎症性疾病，主要由特异性免疫球蛋白 E （IgE） 介导1,2。它的特点是打喷嚏、流鼻涕、鼻塞和鼻痒等症状。随着工业化和城市化，AR的流行率逐渐增加，影响了全球约10%-20%的人口1。免疫荧光 （IF） 技术是一种利用抗体-抗原结合反应的荧光染色方法。它可用于检测和定量特定蛋白质在生物组织或细胞中的分布和表达水平。在AR研究中，IF可以同时检测多个靶点，包括AR相关细胞因子、炎症细胞、受体等，有助于探索AR的发病机制和药物的作用3,4,5,6。
多色免疫荧光 （mIF） 染色过程与传统 IF 非常相似，在每轮染色过程中都增加了一个抗体洗脱步骤。这种修饰能够通过连续的单标记和多轮重新染色同时检测同一组织切片上的多个生物标志物。mIF 基于酪胺信号放大 （TSA），允许重复循环进行 TSA 荧光染色，并使用微波加热去除抗体，同时保留荧光信号 7,8。与传统IF相比，mIF具有以下几个优点：（1）它可以检测传统IF难以识别的弱表达抗原9,10;（2）它提供高质量的染色，并具有更好的信噪比;（3）它允许对组织特异性结构和感兴趣区域进行定量11;（4）多通路多通路有效利用组织，节约有限的病理资源12;（5） 通过 mIF 进行多参数分析，可以更深入地了解组织，揭示隐藏的生物信息13.
总体而言，mIF可以观察同一样品中不同的抗原表达和分布，从而促进靶蛋白的研究。未来，寻求了解多种靶蛋白表达和分布的研究人员将发现这种技术是一个有价值的选择。本研究证明了 mIF 在用 AR 对大鼠鼻粘膜样本进行染色中的应用，并评估了 AR 大鼠模型的建立。
Protocol
实验方案和程序已获得成都中医药大学行政和动物研究委员会的批准（备案号：2022DL-010）。从商业上获得8周龄的雄性Sprague Dawley（SD）大鼠，体重为180-200g（见 材料表），并在受控温度（23±2°C）和相对湿度（55%±10%）的自然光/暗循环下饲养。将12只大鼠随机分为两组：对照组和AR组。所有大鼠都适应了这些条件，并在试验前一周免费获得食物和水。 1. AR大鼠?…
Representative Results
通过OVA腹腔注射和鼻腔激发成功诱导6只SD大鼠进入AR模型。AR组所有大鼠均诱导AR，占该组的100%。AR组所有大鼠均出现打喷嚏、流鼻涕、鼻痒等典型症状。所有行为观察得分≥5分（表2）。 建模第21天H &E染色结果显示，对照大鼠鼻黏膜上皮细胞和纤毛排列良好，无炎症细胞浸润迹象。相反，在AR组中，鼻中隔的粘膜受损并脱落，并伴有明显的中性粒细胞浸润…
Discussion
过敏性鼻炎（AR）是一种非感染性鼻粘膜炎症性疾病，由环境和遗传因素共同引起。它已成为一个全球健康问题，影响工作效率，降低生活质量，损害睡眠、认知功能，并引起烦躁和疲劳。AR 影响着全球 10%-20% 的人口¹，并带来巨大的经济成本，每年在欧盟国家造成高达 30-500 亿欧元的损失18.此外，几项研究已经确定了 AR 与哮喘之间的密切关联18,19,20<sup …
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了四川省科学技术厅（2021YJ0175）的支持。
Materials
| Al(OH)3 | Sollerbauer Biotechnology Co., Ltd | A7130 | |
| 75% ethanol | Anhui Yiren An Co., Ltd | 20210107 | |
| Ammonia | Chengdu Kolon Chemical Co., Ltd | 2021070101 | |
| Anhydrous ethanol | Chengdu Kolon Chemical Co., Ltd | 2022070501 | |
| Anti-fluorescence quenching sealer | SouthernBiotech | 0100-01 | |
| Automatic dyeing machine | Thermo scientific | Varistain Gemini ES | |
| Carrier slides | Nantong Mei Wei De Experimental Equipment Co., Ltd | 220518001 | |
| Citrate-phosphate buffer | Servicebio biotechnology co., Ltd | G1201 | |
| Citric acid antigen repair solution (PH 6.0) | Xavier Biotechnology Co., Ltd | G1201 | |
| Coverslip | Nantong Mei Wei De Experimental Equipment Co. | 220518001 | |
| Coverslip | Nantong Mewtech Life Science Co., Ltd | CS01-2450 | |
| CY3-Tyramide | Sawell Biotechnology Co., Ltd | G1223-50UL | |
| DAPI | Sawell Biotechnology Co., Ltd | G1012 | |
| Decoloring shaker | SCILOGEX | S1010E | |
| EDTA decalcification solution | Wuhan Xavier Biotechnology Co., Ltd | CR2203047 | |
| Electric heating blast dryer | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | |
| Embedding box marking machine | Thermo scientific | PrintMate AS | |
| Embedding machine | Wuhan Junjie Electronics Co., Ltd | JB-P5 | |
| Fast tissue dewatering machine | Thermo scientific | STP420 ES | |
| Film sealer | Thermo scientific | Autostainer 360 | |
| FITC-Tyramide | Sawell Biotechnology Co., Ltd | G1222-50UL | |
| Fluorescence microscope | Sunny Optical Technology Co.Ltd | CX40 | |
| Foxp3 | Affinity Biosciences Co., Ltd | bs-10211R | |
| Freezing table | Wuhan Junjie Electronics Co., Ltd | JB-L5 | |
| Goat Anti-Rabbit IgG H&L (HRP) | Liankebio Co., Ltd | GAR0072 | |
| Goat serum | Biosharp | BL210A | |
| H&E staining kit | Leagene | DH0020 | |
| Hemostatic forceps | Shanghai Medical Devices Co., Ltd | J31010 | |
| Hydrochloric acid | Sichuan Xilong Science Co., Ltd | 210608 | |
| Immunohistochemical pen | Biosharp | BC004 | |
| Microwave oven | Midea | M1-L213B | |
| Neutral gum | Sinopharm Group Chemical Reagent Co., Ltd | 10004160 | |
| Ovalbumin | Sollerbauer Biotechnology Co., Ltd | A804010 | |
| Oven | Shanghai Yiheng Scientific Instruments Co., Ltd | DHG-9240A | |
| Palm centrifuge | SCILOGEX | D1008E | |
| Paraformaldehyde | Beyotime Biotechnology Co., Ltd | P0099-100ml | |
| Pathology section scanner | 3DHISTECH Kft | Pannoramic SCAN | |
| PBS buffer | Biosharp | G4202 | |
| Pipette | Dragon | KE0003087/KA0056573 | |
| Rorγt | Affinity Biosciences Co., Ltd | DF3196 | |
| Scalpel | Quanzhou Excellence Medical Co., Ltd | 20170022 | |
| Self-fluorescent quenching agent Sudan Black B | Bioengineering Co., Ltd | A602008-0025 | |
| Slicer | Thermo scientific | HM325 | |
| Slicing machine | Thermo scientific | HM325 | |
| Slide | Nantong Mewtech Life Science Co., Ltd | PC2-301 | |
| Sprague Dawley rats | Sichuan Academy of Traditional Chinese Medicine | SYX  2023-0100 |
|
| TICAM-1 | Affinity Biosciences Co., Ltd | DF6289 | |
| Tissue scissors | Shanghai Medical Devices Co., Ltd | J22120 | |
| Tissue spreading baking sheet machine | Wuhan Junjie Electronics Co., Ltd | JK-6 | |
| TYR-690 fluorescent dyes | Shanghai Rutron Biotechnology Co., Ltd | RC0086-34RM | |
| Vortex mixer | SCILOGEX | SLK-O3000-S | |
| Water bath-slide drier | Wuhan Junjie Electronics Co., Ltd | JK-6 | |
| Wax trimmer | Wuhan Junjie Electronics Co., Ltd | JXL-818 | |
| Xylene | Chengdu Kolon Chemical Co., Ltd | 2022051901 |
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Cite This Article
Hu, Y., Luo, H., Li, J., Zhang, R., Fu, L., Ren, Q., Chen, Y., Huang, X., Zhou, Z., Yuan, H., Tian, L., Wang, X. Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay. J. Vis. Exp. (199), e65937, doi:10.3791/65937 (2023).