Эта работа была поддержана Департаментом науки и технологий провинции Сычуань (2021YJ0175).

Многоцветная иммунофлюоресценция ткани носа на модели крысы с аллергическим ринитом

<ol>
	<li>Bro&#380;ek, J. L., 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=Allergic+rhinitis+and+its+impact+on+asthma+(ARIA)+Guidelines-2016+revision.">Allergic rhinitis and its impact on asthma (ARIA) Guidelines-2016 revision.</a> The Journal of Allergy and Clinical Immunology. 140 (4), 950-958 (2017).</li><li>Maurer, M., Zuberbier, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undertreatment+of+rhinitis+symptoms+in+Europe:+findings+from+a+cross-sectional+questionnaire+survey.">Undertreatment of rhinitis symptoms in Europe: findings from a cross-sectional questionnaire survey.</a> Allergy. 62 (9), 1057-1063 (2007).</li><li>Ding, Y., 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=Alpha-linolenic+acid+improves+nasal+mucosa+epithelial+barrier+function+in+allergic+rhinitis+by+arresting+CD4++T+cell+differentiation+via+IL-4R&#945;-JAK2-STAT3+pathway.">Alpha-linolenic acid improves nasal mucosa epithelial barrier function in allergic rhinitis by arresting CD4+ T cell differentiation via IL-4R&#945;-JAK2-STAT3 pathway.</a> Phytomedicine: International Journal of Phytotherapy and Phytopharmacology. 116, 154825 (2023).</li><li>Zhou, H., 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=Activation+of+NLRP3+inflammasome+contributes+to+the+inflammatory+response+to+allergic+rhinitis+via+macrophage+pyroptosis.">Activation of NLRP3 inflammasome contributes to the inflammatory response to allergic rhinitis via macrophage pyroptosis.</a> International Immunopharmacology. 110, 109012 (2022).</li><li>Watts, A. M., Cripps, A. W., West, N. P., Cox, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+allergic+inflammation+in+the+nasal+mucosa+of+allergic+rhinitis+sufferers+with+topical+pharmaceutical+agents.">Modulation of allergic inflammation in the nasal mucosa of allergic rhinitis sufferers with topical pharmaceutical agents.</a> Frontiers in pharmacology. 10, 294 (2019).</li><li>Patel, N., 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=Fungal+extracts+stimulate+solitary+chemosensory+cell+expansion+in+noninvasive+fungal+rhinosinusitis.">Fungal extracts stimulate solitary chemosensory cell expansion in noninvasive fungal rhinosinusitis.</a> International Forum of Allergy &amp; Rhinology. 9 (7), 730-737 (2019).</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>Badoua, 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-ex+pressing+tumor-infiltrating+T+cells+are+a+favorable+prognostic+biomarker+in+HPV-associated+head+and+neck+cancer.">PD-1-ex pressing 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>Fedchenko, N., Reifenrath, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+approaches+for+interpretation+and+reporting+of+immunohistochemistry+analysis+results+in+the+bone+tissue+-+a+review.">Different approaches for interpretation and reporting of immunohistochemistry analysis results in the bone tissue - a review.</a> Diagnostic Pathology. 9, 221 (2014).</li><li>Faget, L., Hnasko, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyramide+signal+amplification+for+immunofluorescent+enhancement.">Tyramide signal amplification for immunofluorescent enhancement.</a> Methods in Molecular Biology. 1318, 161-172 (2015).</li><li>Parra, E. R., Villalobos, P., Rodriguez-Canales, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multiple+faces+of+programmed+cell+death+ligand+1+expression+in+malignant+and+nonmalignant+cells.">The multiple faces of programmed cell death ligand 1 expression in malignant and nonmalignant cells.</a> Applied Immunohistochemistry &amp; Molecular Morphology.: AIMM. 27 (4), 287-294 (2019).</li><li>Welsh, A. W., 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=Standardization+of+estrogen+receptor+measurement+in+breast+cancer+suggests+false-negative+results+are+a+function+of+threshold+intensity+rather+than+percentage+of+positive+cells.">Standardization of estrogen receptor measurement in breast cancer suggests false-negative results are a function of threshold intensity rather than percentage of positive cells.</a> Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 29 (22), 2978-2984 (2011).</li><li>Parra, E. R., 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=Validation+of+multiplex+immunofluorescence+panels+using+multispectral+microscopy+for+immune-profiling+of+formalin-fixed+and+paraffin-embedded+human+tumor+tissues.">Validation of multiplex immunofluorescence panels using multispectral microscopy for immune-profiling of formalin-fixed and paraffin-embedded human tumor tissues.</a> Scientific Reports. 7 (1), 13380 (2017).</li><li>Miao, M., Xiang, L., Miao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+for+preparation+of+allergic+rhinitis+animal+model+(draft).">Specification for preparation of allergic rhinitis animal model (draft).</a> Chinese Traditional and Herbal Drugs. 49 (1), 50-57 (2018).</li><li>Xu, J., 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=Astragalus+polysaccharides+attenuate+ovalbumin-induced+allergic+rhinitis+in+rats+by+inhibiting+nlrp3+inflammasome+activation+and+nod2-mediated+nf-+&#954;+b+activation.">Astragalus polysaccharides attenuate ovalbumin-induced allergic rhinitis in rats by inhibiting nlrp3 inflammasome activation and nod2-mediated nf- &#954; b activation.</a> Journal of Medicinal Food. 24 (1), 1-9 (2021).</li><li>Bousquet, J., 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=Allergic+rhinitis.">Allergic rhinitis.</a> Nature reviews. 6 (1), 95 (2020).</li><li>Huang, 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=Expression+of+activated+signal+transducer+and+activator+of+transcription-3+as+a+predictive+and+prognostic+marker+in+advanced+esophageal+squamous+cell+carcinoma.">Expression of activated signal transducer and activator of transcription-3 as a predictive and prognostic marker in advanced esophageal squamous cell carcinoma.</a> World Journal of Surgical Oncology. 13, 314 (2015).</li><li>Leynaert, B., Bousquet, J., Neukirch, C., Liard, R., Neukirch, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perennial+rhinitis:+An+independent+risk+factor+for+asthma+in+nonatopic+subjects:+results+from+the+European+Community+Respiratory+Health+Survey.">Perennial rhinitis: An independent risk factor for asthma in nonatopic subjects: results from the European Community Respiratory Health Survey.</a> The Journal of Allergy and Clinical Immunology. 104 (2), 301-304 (1999).</li><li>Guerra, 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=Rhinitis+is+an+independent+risk+factor+for+developing+cough+apart+from+colds+among+adults.">Rhinitis is an independent risk factor for developing cough apart from colds among adults.</a> Allergy. 60 (3), 343-349 (2005).</li><li>Greisner, W. A., Settipane, R. J., Settipane, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-existence+of+asthma+and+allergic+rhinitis:+a+23-year+follow-up+study+of+college+students.">Co-existence of asthma and allergic rhinitis: a 23-year follow-up study of college students.</a> Allergy and Asthma Proceedings. 19 (4), 185-188 (1998).</li><li>Pawankar, R., Mori, S., Ozu, C., Kimura, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+on+the+pathomechanisms+of+allergic+rhinitis.">Overview on the pathomechanisms of allergic rhinitis.</a> Asia Pacific Allergy. 1 (3), 157-167 (2011).</li><li>Van Nguyen, T., 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=Anti-allergic+rhinitis+activity+of+&#945;-lipoic+acid+via+balancing+th17/treg+expression+and+enhancing+nrf2/ho-1+pathway+signaling.">Anti-allergic rhinitis activity of &#945;-lipoic acid via balancing th17/treg expression and enhancing nrf2/ho-1 pathway signaling.</a> Scientific Reports. 10 (1), 12528 (2020).</li><li>Ivanov, I. 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>

Авторам нечего раскрывать.

Аллергический ринит (АР) – это неинфекционное воспалительное заболевание слизистой оболочки носа, возникающее в результате сочетания экологических и генетических факторов. Это стало глобальной проблемой здравоохранения, влияя на эффективность работы, снижая качество жизни, ухудшая ...

Аллергический ринит (АР) – это хроническое неинфекционное воспалительное заболевание слизистой оболочки носа, преимущественно опосредованное специфическим иммуноглобулином Е (IgE)1,2. Для него характерны такие симптомы, как чихание, насморк, заложенность носа и зуд в носу. По мере индустриализации и урбанизации распространенность АР постепенно увеличивается, затрагивая примерно 10–20% населения мира1. Метод иммунофлуоресценции (ИФ) представляет собой метод флуоресцентного окрашивания, в котором используется реакция связывания антитело-антиген. Он может быть использован для обнаружения и количественной оценки распределения и уровней экспрессии конкретных белков в биологических тканях или клетках. В исследованиях АР ИФ может одновременно обнаруживать несколько мишеней, включая цитокины, связанные с АР, воспалительные клетки, рецепторы и многое другое, что облегчает изучение патогенеза АР и эффектов лекарств 3,4,5,6.
Процесс многоцветного иммунофлуоресцентного окрашивания (mIF) очень похож на традиционный IF, с добавлением этапа элюирования антител во время каждого раунда окрашивания. Эта модификация позволяет одновременно обнаруживать несколько биомаркеров на одном и том же участке ткани с помощью последовательного одиночного мечения и нескольких циклов повторного окрашивания. mIF основан на усилении тирамидного сигнала (TSA), что позволяет проводить повторяющиеся циклы флуоресцентного окрашивания TSA и использовать микроволновый нагрев для удаления антител с сохранением флуоресцентных сигналов 7,8. По сравнению с обычным ИФ, mIF имеет ряд преимуществ: (1) он может обнаруживать слабоэкспрессируемые антигены, которые трудно идентифицировать с помощью обычного IF 9,10; (2) обеспечивает высококачественное окрашивание с улучшенным соотношением сигнал/шум; (3) она позволяет количественно оценить тканеспецифические структуры и области, представляющие интерес11; (4) мультиплексирование нескольких путей эффективно использует ткани и сохраняет ограниченные патологические ресурсы12; (5) многопараметрический анализ с помощью mIF позволяет глубже проникнуть в ткани, выявляя скрытую биологическую информацию13.
В целом, mIF позволяет наблюдать различные экспрессии и распределения антигенов в одном и том же образце, облегчая изучение белков-мишеней. В будущем исследователи, стремящиеся понять экспрессию и распределение нескольких белков-мишеней, найдут этот метод ценным выбором. В данном исследовании демонстрируется применение мИФ для окрашивания образцов слизистой оболочки носа крыс с АР и оценивается создание модели АР на крысах.

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			<td></td>
		</tr>
		<tr>
			<td>FITC-Tyramide</td>
			<td>Sawell Biotechnology Co., Ltd</td>
			<td>G1222-50UL</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Sunny Optical Technology Co.Ltd</td>
			<td>CX40</td>
			<td></td>
		</tr>
		<tr>
			<td>Foxp3</td>
			<td>Affinity Biosciences Co., Ltd</td>
			<td>bs-10211R</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing table</td>
			<td>Wuhan Junjie Electronics Co., Ltd</td>
			<td>JB-L5</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG H&#38;L (HRP)</td>
			<td>Liankebio Co., Ltd</td>
			<td>GAR0072</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Biosharp</td>
			<td>BL210A</td>
			<td></td>
		</tr>
		<tr>
			<td>H&#38;E staining kit</td>
			<td>Leagene</td>
			<td>DH0020</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemostatic forceps</td>
			<td>Shanghai Medical Devices Co., Ltd</td>
			<td>J31010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sichuan Xilong Science Co., Ltd</td>
			<td>210608</td>
			<td></td>
		</tr>
		<tr>
			<td>Immunohistochemical pen</td>
			<td>Biosharp</td>
			<td>BC004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave oven</td>
			<td>Midea</td>
			<td>M1-L213B</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutral gum</td>
			<td>Sinopharm Group Chemical Reagent Co., Ltd</td>
			<td>10004160</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin&#160;</td>
			<td>Sollerbauer Biotechnology Co., Ltd</td>
			<td>A804010</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Shanghai Yiheng Scientific Instruments Co., Ltd</td>
			<td>DHG-9240A</td>
			<td></td>
		</tr>
		<tr>
			<td>Palm centrifuge</td>
			<td>SCILOGEX</td>
			<td>D1008E</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Beyotime Biotechnology Co., Ltd</td>
			<td>P0099-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Pathology section scanner</td>
			<td>3DHISTECH Kft</td>
			<td>Pannoramic SCAN&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Biosharp</td>
			<td>G4202</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette&#160;</td>
			<td>Dragon</td>
			<td>KE0003087/KA0056573</td>
			<td></td>
		</tr>
		<tr>
			<td>Ror&#947;t</td>
			<td>Affinity Biosciences Co., Ltd</td>
			<td>DF3196</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Quanzhou Excellence Medical Co., Ltd</td>
			<td>20170022</td>
			<td></td>
		</tr>
		<tr>
			<td>Self-fluorescent quenching agent Sudan Black B</td>
			<td>Bioengineering Co., Ltd</td>
			<td>A602008-0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicer</td>
			<td>Thermo scientific</td>
			<td>HM325</td>
			<td></td>
		</tr>
		<tr>
			<td>Slicing machine</td>
			<td>Thermo scientific</td>
			<td>HM325</td>
			<td></td>
		</tr>
		<tr>
			<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>
		</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

Аллергический ринит (АР) – это хроническое неинфекционное воспалительное заболевание слизистой оболочки носа, в основном опосредованное специфическим иммуноглобулином Е (IgE), поражающее примерно 10-20% населения мира. В то время как иммунофлуоресцентное (ИФ) окрашивание уже давно является стандартным методом обнаружения специфической для заболевания экспрессии белков, традиционные методы ИФ ограничены в своей способности обнаруживать уровни экспрессии трех или более белков в одном образце. В связи с этим в последние годы были разработаны многоцветные методы ПЧ, которые позволяют одновременно маркировать несколько мишеней в клетках или тканях.
В этом протоколе представлен всесторонний обзор процесса создания модели АР на крысах, получения образцов слизистой оболочки носа и технических процедур многоцветной иммунофлюоресценции. Все крысы в группе AR демонстрировали типичные симптомы, такие как чихание, насморк и зуд в носу, при этом поведенческие наблюдения набрали ≥5 балла. Окрашивание гематоксилином и эозином (H&E) выявило увеличение количества воспалительных клеток и нарушение целостности слизистой оболочки носа в группе АР. Многоцветная иммунофлуоресценция (mIF) демонстрировала повышенную экспрессию RORγt и TICAM-1, в то время как экспрессия Foxp3 снижалась в ткани слизистой оболочки носа крыс с АР.

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.

В центре внимания автора: Продвижение исследований аллергического ринита с помощью многоцветной иммунофлуоресценции

Иммунофлуоресцентное мечение срезов слизистой оболочки носа крыс с аллергическим ринитом методом многоцветного иммуноанализа

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.

Шесть крыс SD были успешно индуцированы в модель AR с помощью внутрибрюшинной инъекции OVA и назального вызова. АР индуцировали у всех крыс в группе АР, что составляет 100% группы. У всех крыс в группе AR наблюдались типичные симптомы, такие как чихание, насморк и зуд в носу. Все поведенческие н?...

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

В данном протоколе описана методика многоцветной иммунофлюоресценции для оценки модели аллергического ринита у крыс.

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

Наше основное исследование основано на изучении аллергического ринита. Наше исследование направлено на то, чтобы проанализировать, какие белки связаны с аллергическим ринитом. Многоцветная иммунофлуоресценция в настоящее время используется для продвижения исследований в нашей области.В будущих исследованиях мы сосредоточимся на возможных механизмах ТКМ в лечении аллергического ринита.

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 Views.  Hospital of Chengdu University of Traditional Chinese Medicine. Наше основное исследование основано на изучении аллергического ринита. Наше исследование направлено на то, чтобы проанализировать, какие белки связаны с аллергическим ринитом. Многоцветная иммунофлуоресценция в настоящее время используется для продвижения исследова...

Video: В центре внимания автора: Продвижение исследований аллергического ринита с помощью многоцветной иммунофлуоресценции

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.

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

Получение и предварительная обработка образцов слизистой оболочки носа у крыс с аллергическим ринитом  

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.

Получение и предварительная обработка образцов слизистой оболочки носа у крыс с аллергическим ринитом

Multicolor Immunostaining of Nasal Mucosal Tissues of Rats with Allergic Rhinitis

Многоцветное иммуноокрашивание тканей слизистой оболочки носа крыс с аллергическим ринитом  

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.