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. 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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">
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</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。

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa primarily mediated by specific immunoglobulin E (IgE)1,2. It is characterized by symptoms such as sneezing, a runny nose, nasal congestion, and nasal itching. With industrialization and urbanization, the prevalence of AR is gradually increasing, affecting approximately 10%-20% of the world&#39;s population1. The immunofluorescence (IF) technique is a fluorescent staining method that utilizes an antibody-antigen binding reaction. It can be employed to detect and quantify the distribution and expression levels of specific proteins in biological tissues or cells. In AR research, IF can simultaneously detect multiple targets, including AR-related cytokines, inflammatory cells, receptors, and more, facilitating the exploration of AR pathogenesis and the effects of drugs3,4,5,6.
The multicolor immunofluorescence (mIF) staining process closely resembles traditional IF, with the addition of an antibody elution step during each round of staining. This modification enables the simultaneous detection of multiple biomarkers on the same tissue section through sequential single-labeling and multiple rounds of re-staining. mIF is based on tyramide signal amplification (TSA), allowing for repeated cycles of TSA fluorescent staining and the use of microwave heating to remove antibodies while retaining fluorescent signals7,8. In comparison to conventional IF, mIF offers several advantages: (1) it can detect weakly expressed antigens that are challenging to identify with conventional IF9,10; (2) it provides high-quality staining with an improved signal-to-noise ratio; (3) it allows for the quantification of tissue-specific structures and regions of interest11; (4) multiplexing multiple pathways efficiently utilizes tissues and conserves limited pathological resources12; (5) multi-parameter analysis through mIF offers deeper insights into tissues, uncovering hidden biological information13.
Overall, mIF allows the observation of different antigen expressions and distributions within the same sample, facilitating the study of target proteins. In the future, researchers seeking to understand the expression and distribution of multiple target proteins will find this technique a valuable choice. This study demonstrates the application of mIF for staining nasal mucosa samples from rats with AR and evaluate the establishment of a rat model of AR.

作者聚焦：利用多色免疫荧光推进过敏性鼻炎研究

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

Our main research is based on a study of allergic rhinitis. Our research intends to analyze what proteins are associated with allergic rhinitis. Multicolor immunofluorescence is currently used to advance research in our field.In future studies, we will focus on the possible mechanisms of TCM in the treatment of allergic rhinitis.

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

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。

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

1.1K 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

Allergic rhinitis (AR) is a chronic, non-infectious inflammatory disease of the nasal mucosa, primarily mediated by specific immunoglobulin E (IgE), affecting approximately 10%-20% of the world's population. While immunofluorescence (IF) staining has long been a standard technique for detecting disease-specific protein expression, conventional IF techniques are limited in their ability to detect the expression levels of three or more proteins in the same sample. Consequently, multicolor IF techniques have been developed in recent years, which allow the simultaneous labeling of multiple targets in cells or tissues.
This protocol provides a comprehensive overview of the process for establishing a rat model of AR, obtaining nasal mucosal samples, and the technical procedures for multicolor immunofluorescence. All rats in the AR group exhibited typical symptoms such as sneezing, a runny nose, and an itchy nose, with behavioral observations scoring &#8805;5 points. Hematoxylin and eosin (H&amp;E) staining revealed increased inflammatory cell counts and disrupted nasal mucosal integrity in the AR group. Multicolor immunofluorescence (mIF) demonstrated increased expression of ROR&#947;t and TICAM-1, while Foxp3 expression decreased in the nasal mucosa tissue of AR rats.

This protocol describes a multicolor immunofluorescence technique for evaluating the rat model of allergic rhinitis.

科研

医学

Acquisition and Pre-Processing of Nasal Mucosal Samples from Rats with Allergic Rhinitis

To begin, with a scalpel, make a precise incision along the two corners of the mouth of a euthanized rat to expose the muscle tissue. Use a pair of tissue scissors to cut the joints of the zygomatic bone and the mandible on both sides of the skull. Then remove the mandible.Use tissue scissors to cut the bony connection between the nasal cavity and the maxilla. Then use a hemostat to separate the skin of the maxilla to expose the nasal cavity. Sever the connection between the nasal cavity and the orbital bones on both sides at the infraorbital foramen, and remove the nasal cavity.Place the extracted nasal cavity in a 4%Paraformaldehyde solution for fixation. To decalcify the tissue, wash the fixed tissue with distilled water. Place the tissues in a decalcification solution with a volume 20 to 30 times that of the tissues at room temperature.Once decalcification is complete, place the tissue in a dehydration box. Transfer the dehydration box to a dehydrator and dehydrate the tissue in increasing alcohol concentrations at different incubation times. Next, place the tissue in anhydrous ethanol for 30 minutes for two cycles.Following this, transfer the tissue to xylene for five to 10 minutes and repeat, then immerse the tissue in wax for one hour. Place the wax block in embedding cassettes. Remove the wax blocks after they have solidified and trim the wax block.Insert the trimmed wax block into a paraffin microtome and cut it into three micrometer thick slices. Hematoxylin and eosin staining of the control rats showed well arranged and cilia. The nasal septum mucosa was damaged and detached in the disease group with notable neutrophil infiltration.

过敏性鼻炎大鼠鼻粘膜样本的采集和预处理

Multicolor Immunostaining of Nasal Mucosal Tissues of Rats with Allergic Rhinitis

To begin, place the hematoxylin and eosin stained rat nasal mucosal tissue sections onto a microscope stage. Adjust the microscope's focus until the image is clearly visible. Capture the tissue image at 40X.Heat the citrate phosphate buffer until it boils. Then add the slides to the container after removing it from the heat. After heating for eight minutes until boiling, turn off the heat for eight minutes and subsequently switch to medium low heat for seven minutes.After allowing the container to cool naturally transfer the slices to PBS. Use a decolorizing shaker to shake the slices for five minutes. Next draw a circle around the dried tissue with an immunohistochemical pen.Add 3%hydrogen peroxide solution to the slices before incubating. Then transfer the slices to PBS for washing as before. To block, apply 5%goat serum within the marked circle.After incubation, carefully remove the blocking solution and add one to two milliliters of FOXP3 to the tissue slices. Then place the slices in a humid chamber for overnight incubation. After washing the slices in PBS, gently dab the slices dry with filter paper.Then add one to two milliliters of the secondary antibody solution to the slices. Next incubate the slices in 100 microliters of tyramide signal amplification working solution for 10 minutes at room temperature in the dark. After incubation, wash the slices with PBS three times for five minutes each.Now apply one to two milliliters of DAPI staining solution to the slices before incubation. Quench the auto fluorescence by incubating the slices in the quencher for five minutes before a 10 minute PBS wash. Then seal them with the anti fluorescence quencher.Capture the microscopic images of the stained sections under 20X and 40X magnification. Hematoxylin and eosin staining of the control rats showed well arranged epithelial cells and cilia. The nasal septum mucosa was damaged and detached in the disease group with notable neutrophil infiltration.Multi immunofluorescent staining revealed that the expressions of ROR Gamma T and TCAM1 were increased in the disease group. However, FOXP3 was under expressed.