Name: Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence
Uploaded: 2023-09-22T14:50:00
Description: Watch this Scientific Journal Video about Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence at JoVE.com

Bu çalışma, Sichuan Eyaleti Bilim ve Teknoloji Departmanı (2021YJ0175) tarafından desteklenmiştir.

Deneysel protokol ve prosedürler, Chengdu Geleneksel Çin Tıbbı Üniversitesi İdari ve Hayvan Araştırma Komitesi'nden onay almıştır (Kayıt numarası: 2022DL-010). 180-200 g ağırlığındaki sekiz haftalık erkek Sprague Dawley (SD) sıçanları ticari olarak elde edildi (Malzeme Tablosuna bakınız) ve kontrollü sıcaklık (23 ± 2 °C) ve bağıl nem (%55 ± %10) ile doğal bir aydınlık/karanlık döngüsü altında barındırıldı. On iki sıçan rastgele iki gruba ayrıldı: kontrol gr...

Multicolor Immunofluorescence of the Nasal Tissue in an Allergic Rhinitis Rat Model

<ol>
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Allerjik Rinit (AR), çevresel ve genetik faktörlerin bir kombinasyonundan kaynaklanan burun mukozasının enfeksiyöz olmayan inflamatuar bir hastalığıdır. İş verimliliğini etkileyen, yaşam kalitesini düşüren, uykuyu, bilişsel işlevi bozan, sinirlilik ve yorgunluğa neden olan küresel bir sağlık sorunu haline geldi. AR, dünya nüfusunun %10-20'sini¹ etkilemekte ve önemli ekonomik maliyetler taşıyarak AB ülkelerinde yıllık 30-50 milyar avroya varan kayıplara neden olmaktadır18...

Allerjik rinit (AR), primer olarak spesifik immünoglobulin E (IgE)1,2'nin aracılık ettiği burun mukozasının kronik, enfeksiyöz olmayan inflamatuar bir hastalığıdır. Hapşırma, burun akıntısı, burun tıkanıklığı ve burun kaşıntısı gibi semptomlarla karakterizedir. Sanayileşme ve kentleşme ile birlikte, AR prevalansı giderek artmakta ve dünya nüfusunun yaklaşık %10-20'sini etkilemektedir1. İmmünofloresan (IF) tekniği, bir antikor-antijen bağlanma reaksiyonu kullanan bir floresan boyama yöntemidir. Biyolojik dokularda veya hücrelerde spesifik proteinlerin dağılımını ve ekspresyon seviyelerini tespit etmek ve ölçmek için kullanılabilir. AR araştırmasında IF, AR ile ilişkili sitokinler, inflamatuar hücreler, reseptörler ve daha fazlası dahil olmak üzere birden fazla hedefi aynı anda tespit edebilir ve AR patogenezinin ve ilaçların etkilerininaraştırılmasını kolaylaştırır 3,4,5,6.
Çok renkli immünofloresan (mIF) boyama işlemi, her boyama turu sırasında bir antikor elüsyon adımının eklenmesiyle geleneksel IF'ye çok benzer. Bu modifikasyon, sıralı tek etiketleme ve çoklu yeniden boyama turları yoluyla aynı doku kesitinde birden fazla biyobelirteçin aynı anda saptanmasını sağlar. mIF, tiramid sinyal amplifikasyonuna (TSA) dayanır ve tekrarlanan TSA floresan boyama döngülerine ve floresan sinyallerinikorurken antikorları uzaklaştırmak için mikrodalga ısıtmanın kullanılmasına izin verir 7,8. Konvansiyonel IF ile karşılaştırıldığında, mIF çeşitli avantajlar sunar: (1) konvansiyonel IF 9,10 ile tanımlanması zor olan zayıf eksprese edilmiş antijenleri tespit edebilir; (2) geliştirilmiş sinyal-gürültü oranı ile yüksek kaliteli boyama sağlar; (3) dokuya özgü yapıların ve ilgilenilen bölgelerin nicelleştirilmesine izin verir11; (4) çoklu yolakların çoğullanması, dokuları verimli bir şekilde kullanır ve sınırlı patolojik kaynakları korur12; (5) mIF aracılığıyla çok parametreli analiz, gizli biyolojik bilgileri ortaya çıkararak dokular hakkında daha derin bilgiler sunar13.
Genel olarak, mIF, aynı numune içinde farklı antijen ekspresyonlarının ve dağılımlarının gözlemlenmesine izin vererek hedef proteinlerin incelenmesini kolaylaştırır. Gelecekte, çoklu hedef proteinlerin ekspresyonunu ve dağılımını anlamak isteyen araştırmacılar bu tekniği değerli bir seçim olarak göreceklerdir. Bu çalışma, sıçanlardan alınan nazal mukoza örneklerinin AR ile boyanması için mIF'nin uygulanmasını göstermekte ve bir AR sıçan modelinin oluşturulmasını değerlendirmektedir.

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			<td>Fast tissue dewatering machine</td>
			<td>Thermo scientific</td>
			<td>STP420 ES</td>
			<td></td>
		</tr>
		<tr>
			<td>Film sealer</td>
			<td>Thermo scientific</td>
			<td>Autostainer 360</td>
			<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

Allerjik rinit (AR), dünya nüfusunun yaklaşık %10-20'sini etkileyen, primer olarak spesifik immünoglobulin E (IgE) aracılık eden, burun mukozasının kronik, enfeksiyöz olmayan inflamatuar bir hastalığıdır. İmmünofloresan (IF) boyama, hastalığa özgü protein ekspresyonunu tespit etmek için uzun süredir standart bir teknik olsa da, geleneksel IF teknikleri, aynı numunedeki üç veya daha fazla proteinin ekspresyon seviyelerini tespit etme yeteneklerinde sınırlıdır. Sonuç olarak, son yıllarda, hücrelerde veya dokularda birden fazla hedefin aynı anda etiketlenmesine izin veren çok renkli IF teknikleri geliştirilmiştir.
Bu protokol, bir AR sıçan modeli oluşturma, nazal mukozal örneklerin alınması ve çok renkli immünofloresan için teknik prosedürler için kapsamlı bir genel bakış sağlar. AR grubundaki tüm sıçanlar hapşırma, burun akıntısı ve burun kaşıntısı gibi tipik semptomlar sergiledi ve davranışsal gözlemler ≥5 puan aldı. Hematoksilen ve eozin (H&E) boyama, AR grubunda artmış inflamatuar hücre sayısı ve bozulmuş nazal mukozal bütünlük ortaya çıkardı. Çok renkli immünofloresan (mIF), RORγt ve TICAM-1 ekspresyonunun arttığını gösterirken, AR sıçanlarının burun mukoza dokusunda Foxp3 ekspresyonu azaldı.

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.

Author Spotlight: Advancing Allergic Rhinitis Research with Multicolor Immunofluorescence

Allerjik Rinit Sıçanlarının Nazal Mukoza Dokusu Kesitlerinde Çok Renkli İmmünoassay ile İmmünofloresan Etiketleme

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.

Altı SD sıçan, OVA intraperitoneal enjeksiyon ve nazal zorlama yoluyla AR modeline başarılı bir şekilde indüklendi. AR, AR grubundaki tüm sıçanlarda indüklendi ve grubun% 100'ünü oluşturdu. AR grubundaki tüm sıçanlar hapşırma, burun akıntısı ve burun kaşıntısı gibi tipik semptomlar sergiledi. Tüm davranışsal gözlemler ≥5 puan aldı (Tablo 2).
Modellemenin 21. günündeki H & E boyama sonuçları, kontrol sıçanlarında, burun mukoza...

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

Bu protokol, alerjik rinitin sıçan modelini değerlendirmek için çok renkli bir immünofloresan tekniğini açıklar.

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

Ana araştırmamız alerjik rinit çalışmasına dayanmaktadır. Araştırmamız, hangi proteinlerin alerjik rinit ile ilişkili olduğunu analiz etmeyi amaçlamaktadır. Çok renkli immünofloresan şu anda alanımızdaki araştırmaları ilerletmek için kullanılmaktadır.Gelecekteki çalışmalarda, alerjik rinit tedavisinde TCM'nin olası mekanizmalarına odaklanacağız.