这项工作得到了四川省科学技术厅（2021YJ0175）的支持。

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大鼠模型的建立
<ol><li>制备致敏溶液:将 30 mg 卵清蛋白 （OVA） 和 3 g Al（OH）3 悬浮在 100 mL 盐水中（见材料表）14,15。</li>
	<li>基本致敏:抓住并握住完全清醒的大鼠，使其腹部朝上。用75%酒精浸泡的棉球对腹部进行消毒。使用1.5mL注射器将步骤1.1中制备的1mL敏化溶液注射到AR组大鼠的左腹腔中。每两天重复一次注射，持续14天（图1A）。 注意: 确保大鼠的头部低于尾巴的位置，以防止插入注射器时对大肠和小肠造成损伤。注射部位位于距腹侧中线 1.5 厘米处。对照组大鼠应每两天腹腔注射一次1mL生理盐水，持续14天。</li>
	<li>鼻腔激发:在基础致敏后的第二天，使用100μL移液管（图1B）通过滴鼻液给予50μL5%OVA，重复此过程7天（图1C）。 注意:通过将 5 g OVA 与 100 mL 盐水混合来制备 5% OVA 溶液。对于对照组，给予相同体积的生理盐水滴剂。</li>
</ol>2.大鼠的行为评分
<ol><li>在完成最后的鼻腔挑战后30分钟内，根据 表1 中概述的评分标准，使用数码相机观察并记录大鼠，以识别诸如鼻子抓挠，打喷嚏和流鼻涕等症状（图1D）。 注意:AR 建模成功的确认基于总分达到 ≥5 分14。</li>
</ol>3. 鼻粘膜样本的采集
<ol><li>牺牲大鼠:通过将大鼠暴露于过量的 CO2 来牺牲大鼠。之后，用75%乙醇对大鼠进行消毒（见 材料表）。用手术刀沿着嘴的两个角做一个切口，露出肌肉组织（图2A）。接下来，使用组织剪刀（图2B）切开颅骨两侧的颧骨和下颌骨的关节，并切除下颌骨（图2C）。</li>
	<li>鼻腔暴露:使用止血器（图2D）分离上颌骨的皮肤以暴露鼻腔（图2E）。用组织剪刀剪断鼻腔和上颌骨之间的骨连接（图2F）。然后，在眶下孔处切断鼻腔和眶骨两侧的连接（图2G）并切除鼻腔（图2H）。</li>
	<li>固定:将提取的鼻腔置于4%多聚甲醛中进行固定，并在室温下储存24-72小时（见 材料表）。 注意: 确保使用的多聚甲醛固定剂的量足以完全覆盖切片以进行正确固定。</li>
</ol>4. 鼻粘膜样本的预处理
<ol><li>脱钙:首先固定步骤3.1中去除的组织。用PBS清洗三次，每次20分钟。然后用蒸馏水清洗三次，每次20分钟。随后，在室温下体积为组织20-30倍的脱钙溶液中对组织进行脱钙。<ol><li>脱钙溶液（见 材料表）应保持7.2-7.4的pH值。最后，将脱钙组织放入脱水盒中。 注意:脱钙溶液需要每周更换一次。当组织软化（约2个月）时，脱钙过程可以结束。</li>
	</ol></li>
	<li>脱水和打蜡:将脱水箱转移到脱水机中（见 材料表）。从75%酒精开始4小时，然后是85%酒精2小时，90%酒精2小时，95%酒精1小时。<ol><li>随后，将组织浸入无水乙醇中30分钟，再重复该过程30分钟，使用二甲苯5-10分钟，再重复此步骤5-10分钟，将组织浸入蜡中1小时，重复此步骤再一个小时，最后，将组织浸入蜡中再浸入1小时。</li>
	</ol></li>
	<li>包埋:将熔化的蜡块放入包埋盒中（见 材料表），并将其储存在-20°C冰箱中。蜡凝固后，将其取出并修剪蜡块。</li>
	<li>切片:将修剪好的蜡块插入石蜡切片机（见 材料表）并将其切成厚度为 3 μm 的切片。将切片置于载玻片撒布器上的40°C水浴中。将纸巾压平，然后用载玻片将其提起，并在60°C烤箱中烘烤。<ol><li>水蒸发后，蜡正确凝固，取出载玻片并将其储存在25°C的温度下。</li>
	</ol></li>
</ol>5. 鼻粘膜组织的H&E染色
<ol><li>脱蜡和水合:将切片置于二甲苯中20分钟，再重复该过程20分钟，无水乙醇5分钟，再重复此步骤5分钟，75%酒精5分钟，最后洗涤5分钟。</li>
	<li>苏木精染色:使用100μL移液管分配100μL苏木精染色液（见 材料表）3-5分钟，冲洗5-10秒，加入100μL0.5%盐酸乙醇10-30秒，冲洗5-10秒，加入100μL0.2%氨水10-30秒，冲洗30秒。 注意:苏木精染色后，需要用0.5%盐酸乙醇进行分化，以去除与细胞核结合并被细胞质吸收的过量苏木精染料。当进行伊红染色时，细胞核和细胞质将被清晰染色。用0.5%盐酸乙醇分化后，切片会出现红色或粉红色，因此分化后立即冲洗以去除组织切片中的酸以阻止分化。然后，使用0.2%氨水增强核染色。</li>
	<li>曙红染色:使用100μL移液管分配100μL曙红染色溶液（参见 材料表）5分钟，然后冲洗30秒。</li>
	<li>脱水和密封:将切片浸入无水乙醇中5分钟，再重复该过程5分钟，再次重复5分钟，二甲基5分钟，二甲苯5分钟。最后，用中性树脂密封（见 材料表）。</li>
	<li>将染色切片放在显微镜上:将染色切片放在显微镜上，调整显微镜的焦距直到图像清晰可见，将放大倍率设置为40倍，然后捕捉图像。</li>
</ol>6.鼻粘膜组织的多色免疫染色
<ol><li>脱蜡和水合作用:遵循步骤 5.1 中提到的相同。</li>
	<li>柠檬酸盐-磷酸盐缓冲液处理:将柠檬酸盐-磷酸盐缓冲液（pH 6.0）（见 材料表）放入微波炉安全容器中。将其加热至沸腾，将其取出，然后将载玻片添加到容器中。用中火微波炉加热8分钟至沸腾，关火8分钟，然后改用中低火加热7分钟。<ol><li>自然冷却后，将切片转移到PBS（pH 7.4）中，摇匀，然后使用脱色摇床洗涤三次，每次5分钟。 注意: 确保缓冲液在此过程中不会蒸发，并防止切片变干。</li>
	</ol></li>
	<li>阻断内源性过氧化物酶:干燥切片，用免疫组化笔在组织周围画一个圆圈（以防止抗体流走），并涂抹3%过氧化氢溶液（过氧化氢:纯水=1:9）。<ol><li>在室温下在黑暗中孵育15分钟。自然冷却后，将切片放入PBS（pH 7.4）中，并在脱色摇床上摇动三个循环，每个循环5分钟。</li>
	</ol></li>
	<li>封闭:在圆圈内涂抹 5% 山羊血清并孵育 30 分钟。</li>
	<li>一抗添加:轻轻除去封闭液，切片中加入FOXP3（稀释度:1:200，见 材料表），置于潮湿室中。在4°C孵育过夜。 注意:在潮湿的腔室中加入少量水以防止抗体蒸发。</li>
	<li>添加二抗:将切片置于PBS（pH 7.4）中，并在脱色振荡器上摇动三个周期，每个周期5分钟。轻轻擦干切片，并在圆圈内应用二抗（山羊抗兔IgG H&L，见 材料表）。在室温下在黑暗中孵育50分钟。</li>
	<li>酪胺稀释液的制备:将 100 μL 3% H 2 O2 与100 mL 1x TBST 混合（参见材料表）。</li>
	<li>加入 TSA 工作溶液:将 1 mL 酪胺稀释液与 2 μL CY3-酪胺混合以制备 TSA 工作溶液。向每个切片上100μLTSA工作溶液，并在室温下在黑暗中孵育10分钟。然后，用PBS清洗切片三次，每次5分钟。 注意:立即制备并使用TSA工作溶液，并将其在4°C避光下储存;有效期最长为24小时。</li>
	<li>重复步骤6.2-6.8:切换到1:200稀释的RORγt（FITC荧光染料），然后切换到1:200稀释的TICAM1（TYP620染料）（参见 材料表）。</li>
	<li>DAPI复染细胞核:轻轻摇动切片干燥，涂上DAPI染色液，在室温下避光孵育10分钟。</li>
	<li>淬灭自发荧光:将切片置于PBS（pH 7.4）中，并在脱色振荡器上摇动三个周期，每个周期5分钟。将自发荧光淬灭剂（参见 材料表）应用于切片，等待5分钟，然后洗涤10分钟。</li>
	<li>封闭:将切片置于PBS（pH 7.4）中，并在脱色摇床上摇动三个周期，每个周期5分钟。轻轻摇晃切片以使其干燥，并用抗荧光淬灭剂密封（参见 材料表）。</li>
	<li>显微镜:将染色切片放在显微镜上，调整显微镜的焦点以获得清晰的可见度，将放大倍率设置为20倍和40倍，然后捕获图像。 注意:DAPI 的紫外激发波长为 330-380 nm，发射波长为 420 nm（蓝光）。FITC的激发波长为465-495 nm，发射波长为515-555 nm（绿光）。CY3 的激发波长为 510-560 nm，发射波长为 590 nm（红光）。TYP-690 的激发波长为 630 nm，发射波长为 690 nm（粉红光）。</li>
</ol>

过敏性鼻炎大鼠模型中鼻组织的多色免疫荧光

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I., 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+orphan+nuclear+receptor+ror&#947;t+directs+the+differentiation+program+of+proinflammatory+IL-17++T+helper+cells.">The orphan nuclear receptor ror&#947;t directs the differentiation program of proinflammatory IL-17+ T helper cells.</a> Cell. 126 (6), 1121-1133 (2006).</li><li>Fontenot, J. D., Gavin, M. A., Rudensky, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxp3+programs+the+development+and+function+of+CD4+CD25++regulatory+T+cells.">Foxp3 programs the development and function of CD4+CD25+ regulatory T cells.</a> Nature Immunology. 4 (4), 330-336 (2003).</li><li>Zhou, X., 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=Instability+of+the+transcription+factor+Foxp3+leads+to+the+generation+of+pathogenic+memory+T+cells+in+Vivo.">Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in Vivo.</a> Nature Immunology. 10 (9), 1000-1007 (2009).</li><li>Ziegler, S. F., Buckner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxp3+and+the+regulation+of+Treg/Th17+differentiation.">Foxp3 and the regulation of Treg/Th17 differentiation.</a> Microbes and Infection. 11 (5), 594-598 (2009).</li><li>Yaling, Y., Gui, Y., Shuqi, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+research+advances+of+toll-like+receptors+and+allergic+rhinitis.">Recent research advances of toll-like receptors and allergic rhinitis.</a> Medical Recapitulate. 23 (7), 1273-1276 (2017).</li><li>Shan, H., Jie, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+TLR4+gene+polymorphisms+and+allergic+rhinitis.">Correlation between TLR4 gene polymorphisms and allergic rhinitis.</a> Journal of Clinical Otorhinolaryngology, Head, and Neck Surgery. 31 (13), 991-994 (2017).</li><li>Xu, 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=Immunoregulatory+mechanism+of+Treg+cells+and+TICAM-1+patients+with+severe+allergic+rhinitis.">Immunoregulatory mechanism of Treg cells and TICAM-1 patients with severe allergic rhinitis.</a> Chinese Journal of Coal Industry Medicine. 23 (3), 303-306 (2020).</li><li>Granier, C., 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=Tim-3+expression+on+tumor-infiltrating+PD-1+CD8++T+cells+correlates+with+poor+clinical+outcome+in+renal+cell+carcinoma.">Tim-3 expression on tumor-infiltrating PD-1+CD8+ T cells correlates with poor clinical outcome in renal cell carcinoma.</a> Cancer Research. 77 (5), 1075-1082 (2017).</li><li>Badoual, C., 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=PD-1+expressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+hpv-associated+head+and+neck+cancer.">PD-1 expressing tumor-infiltrating T cells are a favorable prognostic biomarker in hpv-associated head and neck cancer.</a> Cancer Research. 73 (1), 128-138 (2013).</li><li>Gustavson, M. 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=Development+of+an+unsupervised+pixel-based+clustering+algorithm+for+compartmentalization+of+immunohistochemical+expression+using+automated+quantitative+analysis.">Development of an unsupervised pixel-based clustering algorithm for compartmentalization of immunohistochemical expression using automated quantitative analysis.</a> Applied Immunohistochemistry &amp; Molecular Morphology. 17 (4), 329-337 (2009).</li><li>Lu, S., 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=Comparison+of+biomarker+modalities+for+predicting+response+to+PD-1/PD-L1+checkpoint+blockade:+a+systematic+review+and+meta-analysis.">Comparison of biomarker modalities for predicting response to PD-1/PD-L1 checkpoint blockade: a systematic review and meta-analysis.</a> JAMA Oncology. 5 (8), 1195-1204 (2019).</li><li>Hernandez, S., 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=Multiplex+immunofluorescence+tyramide+signal+amplification+for+immune+cell+profiling+of+paraffin-embedded+tumor+tissues.">Multiplex immunofluorescence tyramide signal amplification for immune cell profiling of paraffin-embedded tumor tissues.</a> Frontiers in Molecular Biosciences. 8, 667067 (2021).</li></ol>

作者没有什么可透露的。

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 ...

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。

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src=&quot;/files/ftp_upload/65937/65937eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt; 2023-0100</td><td></td></tr><tr><td>TICAM-1</td><td>Affinity Biosciences Co., Ltd</td><td>DF6289</td><td></td></tr><tr><td>Tissue scissors</td><td>Shanghai Medical Devices Co., Ltd</td><td>J22120</td><td></td></tr><tr><td>Tissue spreading baking sheet machine</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JK-6</td><td></td></tr><tr><td>TYR-690 fluorescent dyes</td><td>Shanghai Rutron Biotechnology Co., Ltd</td><td>RC0086-34RM</td><td></td></tr><tr><td>Vortex mixer</td><td>SCILOGEX</td><td>SLK-O3000-S</td><td></td></tr><tr><td>Water bath-slide drier</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JK-6</td><td></td></tr><tr><td>Wax trimmer</td><td>Wuhan Junjie Electronics Co., Ltd</td><td>JXL-818</td><td></td></tr><tr><td>Xylene</td><td>Chengdu Kolon Chemical Co., Ltd</td><td>2022051901</td><td></td></tr></tbody></table>

immunofluorescent labeling in nasal mucosa tissue sections of allergic rhinitis rats via multicolor immunoassay

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。

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...

Watch this Scientific Journal Video about Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence at JoVE.com

作者聚焦:利用多色免疫荧光推进过敏性鼻炎研究

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。

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

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), ...

我们的主要研究是基于过敏性鼻炎的研究。我们的研究旨在分析哪些蛋白质与过敏性鼻炎有关。多色免疫荧光目前用于推进我们领域的研究。在未来的研究中，我们将重点关注中医治疗过敏性鼻炎的可能机制。

immunofluorescent-labeling-nasal-mucosa-tissue-sections-allergic

Research

JoVE Journal

Medicine

Immunofluorescent Labeling in Nasal Mucosa Tissue Sections of Allergic Rhinitis Rats via Multicolor Immunoassay

1.5K 次观看.  Hospital of Chengdu University of Traditional Chinese Medicine. 我们的主要研究是基于过敏性鼻炎的研究。我们的研究旨在分析哪些蛋白质与过敏性鼻炎有关。多色免疫荧光目前用于推进我们领域的研究。在未来的研究中，我们将重点关注中医治疗过敏性鼻炎的可能机制。

视频: 作者聚焦:利用多色免疫荧光推进过敏性鼻炎研究

Murine Model of Allergen Induced Asthma

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, with the prevalence increasing.2 As with other diseases, animal models of allergic airway disease greatly facilitate understanding of the underlying pathophysiology, help identify potential therapeutic targets, and allow preclinical testing of possible new therapies. Models of allergic airway disease have been developed in several animal species, but murine models are particularly attractive due to the low cost, ready availability, and well-characterized immune systems of these animals.3 Availability of a variety of transgenic strains further increases the attractiveness of these models.4 Here we describe two murine models of allergic airway disease, both employing ovalbumin as the antigen. Following initial sensitization by intraperitoneal injection, one model delivers the antigen challenge by nebulization, the other by intratracheal delivery. These two models offer complementary advantages, with each mimicking the major features of human asthma.5
The major features of acute asthma include an exaggerated airway response to stimuli such as methacholine (airway hyperresponsiveness; AHR) and eosinophil-rich airway inflammation. These are also prominent effects of allergen challenge in our murine models,5,6 and we describe techniques for measuring them and thus evaluating the effects of experimental manipulation. Specifically, we describe both invasive7 and non-invasive8 techniques for measuring airway hyperresponsiveness as well as methods for assessing infiltration of inflammatory cells into the airways and the lung. Airway inflammatory cells are collected by bronchoalveolar lavage while lung histopathology is used to assess markers of inflammation throughout the organ. These techniques provide powerful tools for studying asthma in ways that would not be possible in humans.

Experimental mouse models of allergic asthma offer new possibilities for studying disease pathogenesis and developing new therapeutics. These models are well suited to measuring factors governing the allergic immune response, airway inflammation, and pulmonary pathophysiology.

过敏原诱发的哮喘小鼠模型

Asthma is a major cause of morbidity and mortality, affecting some 300 million people throughout the world.1 More than 8% of the US population has asthma, ...

科研

免疫与感染

Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3.
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.

We describe a strategy to monitor maturation and migration of pulmonary dendritic cells in response to ovalbumin in the setting of ovalbumin induced allergic airway inflammation. This strategy can be modified to assess migration of pulmonary dendritic cells in settings of infection.

过敏性气道炎症在肺树突状细胞的成熟和迁移分析

Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in ...

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

神经科学

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

引物对免疫冰冻切片大鼠脑组织:例为染色小胶质细胞和神经元

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate (NMDA) Receptor in Blood

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune encephalitis. Detection of the specific autoantibody against the NMDA receptor in the blood or cerebrospinal fluid (CSF) is essential for the accurate diagnosis of this condition. The NMDA receptor is an ion channel protein complex that contains four subunits, including two mandatory NMDA receptor subunit 1 (NR1) and one or two NMDA receptor subunit 2A (NR2A), NMDA receptor subunit 2B (NR2B), NMDA receptor subunit 2C (NR2C), or NMDA receptor subunit 2D (NR2D). The epitope of anti-NMDA receptor autoantibody was reported to be present at the extracellular N-terminal domain of the NR1 subunit of the NMDA receptor. The goal of this study is to develop a simple cell-based immunofluorescence assay that can be used as a screening test to detect the presence of autoantibodies against NR1 subunit of the NMDA receptor in the blood to facilitate the clinical and basic research of anti-NMDA receptor autoimmune encephalitis.

A Simple Cell-based Immunofluorescence Assay to Detect Autoantibody Against the N-Methyl-D-Aspartate NMDA Receptor in Blood

We ectopically expressed NR1 subunit of NMDA receptor tagged with green fluorescent protein in human embryonic cells (HEK293) as antigen to detect autoantibodies against NMDA receptor in the blood of patients suspected with autoimmune encephalitis. This simple method may be suitable for screening purposes in clinical settings.

血液中 n-甲基-d-天门冬氨酸 (NMDA) 受体自身抗体的简易细胞免疫荧光检测

The presence of anti-NMDA receptor autoantibody can cause various neuropsychiatric symptoms in the affected patients, termed anti-NMDA receptor autoimmune ...

Absorption of Nasal and Bronchial Fluids: Precision Sampling of the Human Respiratory Mucosa and Laboratory Processing of Samples

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the human respiratory tract. NA is a non-invasive technique which absorbs fluid from the inferior turbinate, and causes minimal discomfort. NA has yielded reproducible results with the ability to frequently repeat sampling of the upper airway. 
By comparison, alternative methods of sampling the respiratory mucosa, such as nasopharyngeal aspiration (NPA) and conventional swabbing, are more invasive and may result in greater data variability. Other methods have limitations, for instance, biopsies and bronchial procedures are invasive, sputum contains many dead and dying cells and requires liquefaction, exhaled breath condensate (EBC) contains water and saliva, and lavage samples are dilute and variable.
BA can be performed through the working channel of a bronchoscope in clinic. Sampling is well tolerated and can be conducted at multiple sites in the airway. BA results in MLF samples being less dilute than bronchoalveolar lavage (BAL) samples.
This article demonstrates the techniques of NA and BA, as well as the laboratory processing of the resulting samples, which can be tailored to the desired downstream biomarker being measured. These absorption techniques are useful alternatives to the conventional sampling techniques used in clinical respiratory research.

This manuscript describes the use of nasal and bronchial absorption techniques, specifically using synthetic absorptive matrices (SAM) to sample the mucosal lining fluid (MLF) of the upper and lower airway. These methods provide better standardization and tolerability than existing respiratory sampling techniques.

鼻腔和支气管液的吸收: 人体呼吸道黏膜的精确取样和样品的实验室处理

The methods of nasal absorption (NA) and bronchial absorption (BA) use synthetic absorptive matrices (SAM) to absorb the mucosal lining fluid (MLF) of the ...

医学

Identification and Characterization of Immunogenic RNA Species in HDM Allergens that Modulate Eosinophilic Lung Inflammation

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses and microbial substances that induce innate immune responses. These allergen-associated microbial components play an important role in regulating the development of type 2 inflammatory conditions such as allergic asthma. However, the underlying mechanisms remain largely undefined. The protocol presented here determines the structural characteristics and in vivo activity of allergen-associated immunostimulatory RNA. Specifically, common allergens are examined for the presence of double-stranded RNA (dsRNA) species that can stimulate IFN responses in lungs and restrain&#160;the development of severe lung eosinophilia in a mouse model of HDM-induced allergic asthma. Here, we have included the following three assays: Dot blot to show the dsRNA structures in total RNA isolated from allergens including HDM species, RT-qPCR to measure the activities of HDM RNA in interferon stimulating genes (ISGs) expression in mouse lungs and FACS analysis to determine the effects of HDM RNA on the number of eosinophils in BAL and lung, respectively.

Environmental allergens such as house dust mites (HDM) often contain microbial substances that activate innate immune responses to regulate allergic inflammation. The protocol presented here demonstrates the identification of dsRNA species in HDM allergens and characterization of their immunogenic activities in modulating eosinophilic lung inflammation.

HDM过敏原中免疫原RNA物种的识别和特征，可调节嗜酸性肺炎

Environmental allergens such as house dust mites (HDM) are often in complex forms containing both allergic proteins that drive aberrant type 2 responses ...

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to assess the respiratory immune/inflammatory response to this bacterium. Flow cytometry and confocal microscopy are fluorescence-based technologies that allow detailed characterization of biological responses.&#160;Different forms of Hi antigen can be used, including cell wall components, killed/inactivated preparations, and live bacteria. Hi is a fastidious bacterium that requires enriched media but is generally easy to grow in standard laboratory settings. Tissue samples for stimulation with Hi may be obtained from peripheral blood, bronchoscopy, or resected lung (e.g., in patients undergoing surgery for the treatment of lung cancer). Macrophage and neutrophil function may be comprehensively assessed using flow cytometry with a variety of parameters measured, including phagocytosis, reactive oxygen species, and intracellular cytokine production. Lymphocyte function (e.g., T cell and NK cell function) may be specifically assessed using flow cytometry, principally for intracellular cytokine production. Hi infection is a potent inducer of extracellular trap production, both by neutrophils (NETs) and macrophages (METs). Confocal microscopy is arguably the most optimal way to assess NET and MET expression, which may also be used to assess protease activity. Lung immunity to Haemophilus influenzae can be assessed using flow cytometry and confocal microscopy.

Assessing Respiratory Immune Responses to Haemophilus Influenzae

Haemophilus influenzae induces inflammation in the respiratory tract. This article will focus on the use of flow cytometry and confocal microscopy to define immune responses by phagocytes and lymphocytes in response to this bacterium.

评估对流感嗜血杆菌的呼吸道免疫应答

Haemophilus influenzae (Hi) is a prevalent bacterium found in a range of respiratory conditions. A variety of different assays/techniques may be used to ...

Isolation of Group 2 Innate Lymphoid Cells from Mouse Nasal Mucosa to Detect the Expression of CD226

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various pathological processes, including anti-helminth immunity, tissue repair, thermogenesis, and autoimmune diseases such as asthma and allergic rhinitis (AR). ILC2s permanently reside in peripheral tissues such as the skin, gut, lungs, and nasal cavity; however, there is limited information about their exact functions in nasal mucosal immunity. CD226 is an activating costimulatory molecule, mainly expressed on natural killer (NK) cells, T cells, and inflammatory monocytes. However, whether ILC2s express CD226 or play a role in the pathogenesis of ILC2s-related diseases remains unknown. Here, we established a method to isolate and identify ILC2s from the nasal mucosa and detected CD226 expression on ILC2s obtained from healthy and AR mice. Herein, we describe this protocol for the isolation and identification of ILC2s from mouse nasal mucosa, which will help explore the internal pathological mechanism of immunological disorders in nasal mucosal diseases.

Group 2 innate lymphoid cells (ILC2s), implicated in type 2 inflammation, mainly participate in response to helminth infection, allergic diseases, metabolic homeostasis, and tissue repair. In this study, a procedure to isolate ILC2s from murine nasal mucosa and detect the expression of CD226 is demonstrated.

从小鼠鼻粘膜中分离第2组先天淋巴细胞以检测CD226的表达

With abundant research on group 2 innate lymphoid cells (ILC2s) published over the years, ILC2s are widely known to be implicated in regulating various ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

石蜡内嵌组织中人与鼠中性粒细胞外陷阱的免疫荧光标记

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...