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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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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
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			<td>Sollerbauer Biotechnology Co., Ltd</td>
			<td>A7130</td>
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			<td>20210107</td>
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			<td>2021070101</td>
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			<td>SouthernBiotech</td>
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			<td>Thermo scientific</td>
			<td>Varistain Gemini ES</td>
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			<td>Carrier slides</td>
			<td>Nantong Mei Wei De Experimental Equipment Co., Ltd</td>
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			<td>Servicebio biotechnology co., Ltd</td>
			<td>G1201</td>
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			<td>Citric acid antigen repair solution (PH 6.0)</td>
			<td>Xavier Biotechnology Co., Ltd</td>
			<td>G1201</td>
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			<td>Nantong Mei Wei De Experimental Equipment Co.</td>
			<td>220518001</td>
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			<td>Nantong Mewtech Life Science Co., Ltd</td>
			<td>CS01-2450</td>
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			<td>Sawell Biotechnology Co., Ltd</td>
			<td>G1223-50UL</td>
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			<td>Sawell Biotechnology Co., Ltd</td>
			<td>G1012</td>
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			<td>SCILOGEX</td>
			<td>S1010E</td>
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			<td>EDTA decalcification solution</td>
			<td>Wuhan Xavier Biotechnology Co., Ltd</td>
			<td>CR2203047</td>
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			<td>Electric heating blast dryer&#160;</td>
			<td>Shanghai Yiheng Scientific Instruments Co., Ltd</td>
			<td>DHG-9240A</td>
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			<td>Thermo scientific</td>
			<td>&#160;PrintMate AS</td>
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			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-P5</td>
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			<td>Thermo scientific</td>
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			<td>Thermo scientific</td>
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			<td>FITC-Tyramide</td>
			<td>Sawell Biotechnology Co., Ltd</td>
			<td>G1222-50UL</td>
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			<td>Fluorescence microscope</td>
			<td>Sunny Optical Technology Co.Ltd</td>
			<td>CX40</td>
			<td></td>
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			<td>Foxp3</td>
			<td>Affinity Biosciences Co., Ltd</td>
			<td>bs-10211R</td>
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			<td>Freezing table</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-L5</td>
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			<td>Goat Anti-Rabbit IgG H&#38;L (HRP)</td>
			<td>Liankebio Co., Ltd</td>
			<td>GAR0072</td>
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			<td>Biosharp</td>
			<td>BL210A</td>
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			<td>H&#38;E staining kit</td>
			<td>Leagene</td>
			<td>DH0020</td>
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			<td>Hemostatic forceps</td>
			<td>Shanghai Medical Devices Co., Ltd</td>
			<td>J31010</td>
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			<td>Sichuan Xilong Science Co., Ltd</td>
			<td>210608</td>
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			<td>Biosharp</td>
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			<td>Midea</td>
			<td>M1-L213B</td>
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			<td>Sinopharm Group Chemical Reagent Co., Ltd</td>
			<td>10004160</td>
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			<td>Ovalbumin&#160;</td>
			<td>Sollerbauer Biotechnology Co., Ltd</td>
			<td>A804010</td>
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			<td>Shanghai Yiheng Scientific Instruments Co., Ltd</td>
			<td>DHG-9240A</td>
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			<td>Palm centrifuge</td>
			<td>SCILOGEX</td>
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			<td>Paraformaldehyde</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>P0099-100ml</td>
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			<td>3DHISTECH Kft</td>
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			<td>PBS buffer</td>
			<td>Biosharp</td>
			<td>G4202</td>
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			<td>Pipette&#160;</td>
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			<td>KE0003087/KA0056573</td>
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			<td>Ror&#947;t</td>
			<td>Affinity Biosciences Co., Ltd</td>
			<td>DF3196</td>
			<td></td>
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			<td>Quanzhou Excellence Medical Co., Ltd</td>
			<td>20170022</td>
			<td></td>
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			<td>Bioengineering Co., Ltd</td>
			<td>A602008-0025</td>
			<td></td>
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			<td>Slicer</td>
			<td>Thermo scientific</td>
			<td>HM325</td>
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			<td>Slicing machine</td>
			<td>Thermo scientific</td>
			<td>HM325</td>
			<td></td>
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			<td>Slide</td>
			<td>Nantong Mewtech Life Science Co., Ltd</td>
			<td>PC2-301</td>
			<td></td>
		</tr>
		<tr>
			<td>Sprague Dawley rats</td>
			<td>Sichuan Academy of Traditional Chinese Medicine</td>
			<td>SYX <img alt="Equation 1" src="/files/ftp_upload/65937/65937eq01.jpg" style="margin: auto;" /> 2023-0100</td>
			<td></td>
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			<td>TICAM-1</td>
			<td>Affinity Biosciences Co., Ltd</td>
			<td>DF6289</td>
			<td></td>
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			<td>Tissue scissors</td>
			<td>Shanghai Medical Devices Co., Ltd</td>
			<td>J22120</td>
			<td></td>
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			<td>Tissue spreading baking sheet machine</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JK-6</td>
			<td></td>
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			<td>TYR-690 fluorescent dyes</td>
			<td>Shanghai Rutron Biotechnology Co., Ltd</td>
			<td>RC0086-34RM</td>
			<td></td>
		</tr>
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			<td>Vortex mixer</td>
			<td>SCILOGEX</td>
			<td>SLK-O3000-S</td>
			<td></td>
		</tr>
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			<td>Water bath-slide drier</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JK-6</td>
			<td></td>
		</tr>
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			<td>Wax trimmer</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JXL-818</td>
			<td></td>
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		<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

High-definition Fourier Transform Infrared (FT-IR) Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated biochemical information. FT-IR imaging of tissue is based on the principle that different regions of the mid-infrared are absorbed by different chemical bonds (e.g., C=O, C-H, N-H) within cells or tissue that can then be related to the presence and composition of biomolecules (e.g., lipids, DNA, glycogen, protein, collagen). In an FT-IR image, every pixel within the image comprises an entire Infrared (IR) spectrum that can give information on the biochemical status of the cells that can then be exploited for cell-type or disease-type classification. In this paper, we show: how to obtain IR images from human tissues using an FT-IR system, how to modify existing instrumentation to allow for high-definition imaging capabilities, and how to visualize FT-IR images. We then present some applications of FT-IR for pathology using the liver and kidney as examples. FT-IR imaging holds exciting applications in providing a novel route to obtain biochemical information from cells and tissue in an entirely label-free non-perturbing route towards giving new insight into biomolecular changes as part of disease processes. Additionally, this biochemical information can potentially allow for objective and automated analysis of certain aspects of disease diagnosis.

High-definition Fourier Transform Infrared FT-IR Spectroscopic Imaging of Human Tissue Sections towards Improving Pathology

Fourier Transform Infrared (FT-IR) spectroscopic imaging is a fast and label-free approach to obtain biochemical data sets of cells and tissues. Here, we demonstrate how to obtain high-definition FT-IR images of tissue sections towards improving disease diagnosis.

高清傅立叶变换人体组织切片红外（FT-IR）光谱成像对提高病理

High-definition Fourier Transform Infrared (FT-IR) spectroscopic imaging is an emerging approach to obtain detailed images that have associated ...

科研

医学

A Component-resolved Diagnostic Approach for a Study on Grass Pollen Allergens in Chinese Southerners with Allergic Rhinitis and/or Asthma

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen allergens, data on this topic are limited in southern China. Any available data were obtained by self-report questionnaires, skin prick tests, and total or specific IgE tests using crude extracts. For many reasons, these methods are unreliable. Serum sIgE reactivity to Bermuda grass, Timothy grass, and Humulus scandens allergens in a cohort of patients from Greater Guangzhou (southern China&#39;s largest city and its outskirts) with allergic rhinitis and/or asthma were examined using a fully-automated immunoassay analyzer as a component-resolved diagnostics (CRD) tool.
For the first time, a considerably high prevalence of Bermuda grass sIgE positivity was demonstrated in Chinese southerners with allergic rhinitis and/or asthma. In these patients, a subtle prevalence of sensitization to Timothy grass and Humulus scandens was also noted, which may arise from cross-reactivity, as the latter two are not common in the region. This was also supported by the detection of allergen components.
Fully-automated immunoassay analyzers may offer satisfactory consistency between regions, laboratories, and institutions and over time. The automaticity of the instrument may enable a standardized detection that would not have been readily revealed before the advent of CRD.
This is a study that uses a CRD approach to investigate sensitization to grass pollen allergens in southern China. It adds to current evidence in the literature. Future studies are needed to validate these findings. However, although CRD is a useful tool, the findings made with the fully-automated immunoassay analyzer should not substitute for other laboratory investigations, clinical evaluations, and physician expertise.

This work describes a protocol that uses a component-resolved approach to study sensitization to grass pollen allergens in a cohort of patients from southern China with allergic rhinitis and/or asthma.

中国南方与过敏性鼻炎和/或哮喘的花粉过敏原研究的成分分析诊断方法

Sensitization to grass pollen imposes a global risk for allergic airway diseases. Although prevention relies on local investigation of the pollen ...

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 ...

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) function are monitored by the change in voltage across the nasal epithelium after the superfusion of solutions that modify ion channel activity. This is enabled by the measurement of the potential difference between the subcutaneous compartment and the airway epithelium in the nostril, utilizing a catheter in contact with the inferior nasal turbinate.
The test allows the measurement of the stable baseline voltage and the successive net voltage changes after perfusion of 100 &#181;M amiloride, an inhibitor of Na+ reabsorption in Ringer&#39;s solution; a chloride-free solution containing amiloride to drive chloride secretion and 10 &#181;M isoproterenol in a chloride-free solution with amiloride to stimulate the cyclic adenosine monophosphate (cAMP)-dependent chloride conductance related to CFTR.
This technique has the advantage of demonstrating the electrophysiological properties of two key components establishing the hydration of the airway surface liquid of the respiratory epithelium, ENaC, and CFTR. Therefore, it is a useful research tool for phase 2 and proof of concept trials of agents that target CFTR and ENaC activity for the treatment of cystic fibrosis (CF) lung disease. It is also a key follow-up procedure to establish CFTR dysfunction when genetic testing and sweat testing are equivocal. Unlike sweat chloride, the test is relatively more time consuming and costly. It also requires operator training and expertise to conduct the test effectively. Inter- and intra-subject variability has been reported in this technique especially in young or uncooperative subjects. To assist with this concern, interpretation has been improved through a recently validated algorithm.

Standardized Measurement of Nasal Membrane Transepithelial Potential Difference NPD

Here, we present a standardized protocol to measure the nasal potential difference (NPD). Cystic fibrosis transmembrane conductance regulator (CFTR) and epithelial sodium channel (ENaC) function are evaluated by the change in the voltage across the nasal epithelium after superfusion of solutions that modify ion channel activity, providing an outcome measure.

鼻膜 Transepithelial 电位差的标准化测量 (NPD)

We describe a standardized measurement of nasal potential difference (NPD). In this technique, cystic fibrosis transmembrane conductance regulator (CFTR) ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

通过物理和各向异性扩张对人体组织部分的纳米成像

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

Multicolor 3D Printing of Complex Intracranial Tumors in Neurosurgery

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. The model can be used for planning and simulating critical steps of a surgical approach. Therefore, it is important that anatomical structures such as blood vessels inside a tumor can be printed to be colored not only on their surface, but throughout their whole volume. During simulation this allows for the removal of certain parts (e.g., with a high-speed drill) and revealing internally located structures of a different color. Thus, diagnostic information from various imaging modalities (e.g., CT, MRI) can be combined in a single compact and tangible object.
However, preparation and printing of such a fully colored anatomical model remains a difficult task. Therefore, a step-by-step guide is provided, demonstrating the fusion of different cross-sectional imaging data sets, segmentation of anatomical structures, and creation of a virtual model. In a second step the virtual model is printed with volumetrically colored anatomical structures using a plaster-based color 3D binder jetting technique. This method allows highly accurate reproduction of patient-specific anatomy as shown in a series of 3D-printed petrous apex chondrosarcomas. Furthermore, the models created can be cut and drilled, revealing internal structures that allow for simulation of surgical procedures.

The protocol describes the fabrication of fully colored three-dimensional prints of patient-specific, anatomical skull models to be used for surgical simulation. The crucial steps of combining different imaging modalities, image segmentation, three-dimensional model extraction, and production of the prints are explained.

神经外科复杂颅内肿瘤的多色3D打印

Three-dimensional (3D) printing technologies offer the possibility of visualizing patient-specific pathologies in a physical model of correct dimensions. ...

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride (CCl4) Exposure Through an Orogastric Tube

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including complications such as hepatic encephalopathy and impaired protein synthesis. Appropriate animal models are vital to test the mechanism and pathophysiology of ALI and investigate different hepatoprotective strategies. Due to its ability to perform chemical transformations, carbon tetrachloride (CCl4) is widely used in the liver to induce ALI through the formation of reactive oxygen species. CCl4 exposure can be performed intraperitoneally, by inhalation, or through a nasogastric or orogastric tube. Here, we describe a rodent model, in which ALI is induced by CCl4 exposure through an orogastric tube. This method is inexpensive, easily performed, and has minimal hazard risk. The model is highly reproducible and can be widely used to determine the efficacy of potential hepatoprotective strategies and assess markers of liver injury.

Inducing Acute Liver Injury in Rats via Carbon Tetrachloride CCl4 Exposure Through an Orogastric Tube

This protocol describes a common and feasible method of inducing acute liver injury (ALI) via CCl4 exposure through an orogastric tube. CCl4 exposure induces ALI through the formation of reactive oxygen species during its biotransformation in the liver. This method is used to analyze the pathophysiology of ALI and examine different hepatoprotective strategies.

通过四氯化碳诱导大鼠急性肝损伤 （CCl4） 通过奥罗胃管暴露

Acute liver injury (ALI) plays a crucial role in the development of hepatic failure, which is characterized by severe liver dysfunction including ...

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid hormone (PTH), which leads to high serum phosphorus levels and low serum calcium levels. HypoPT most commonly results from accidental damage to the glands or their removal during thyroid or other anterior neck surgery. Parathyroid/thyroid surgery has become more common in recent years, with a corresponding rise in the occurrence of HypoPT as a postoperative complication. There is a critical need for a HypoPT animal model to better understand the mechanisms underlying the effects of HypoPT on mineral ion homeostasis and to verify the therapeutic effectiveness of novel treatments. Here, a technique is reported to create acquired HypoPT in male rats by performing parathyroidectomy (PTX) using carbon nanoparticles. The rat model shows great promise over the mouse models of hypoparathyroidism.&#160;Importantly, the human PTH receptor binding region has an 84.2% sequence similarity with that of the rat, which is higher than the 73.7% similarity shared with mice. Moreover, the effects of estrogen, which can affect the PTH/PTHrP receptor signaling pathway, have not been fully investigated in male rats. Carbon nanoparticles are lymphatic tracers that stain the thyroid lymph nodes black without affecting their function, but they do not stain the parathyroid glands, which makes them easy to identify and remove. In this study, serum PTH levels were undetectable after PTX, and this resulted in significant hypocalcemia and hyperphosphatemia. Thus, the clinical state of postoperative HypoPT can be remarkably represented in the rat model. Carbon-nanoparticle-assisted PTX can, therefore, serve as an extraordinarily effective and readily implementable model for studying the pathogenesis, treatment, and prognosis of HypoPT.

Generation of Hypoparathyroid Rats via Carbon-Nanoparticle-Assisted Parathyroidectomy

An animal model of acquired hypoparathyroidism (HypoPT) is crucial to understanding how HypoPT affects mineral ion homeostasis and to verify the effectiveness of novel treatments. Here, a technique is presented to generate an acquired hypoparathyroidism (AHypoPT) rat model by parathyroidectomy (PTX) using carbon nanoparticles.

通过碳纳米粒子辅助甲状旁腺切除术 产生 甲状旁腺功能减退大鼠

Hypoparathyroidism (HypoPT) is a rare disease involving the parathyroid glands that is characterized by a reduced secretion or potency of the parathyroid ...

使用琼脂糖包埋的振动切片机进行创新的肺部成像

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

作者聚焦:使用琼脂糖包埋的肺组织切片研究空间蛋白表达 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

用于多重荧光成像的琼脂糖包埋肺组织的自动振动切片机切片

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...

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Subtitles

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