Name: Acquisition and Pre-Processing of Nasal Mucosal Samples from Rats with Allergic Rhinitis
Uploaded: 2023-09-22T14:50:00
Description: Watch this Scientific Journal Video about Acquisition and Pre-Processing of Nasal Mucosal Samples from Rats with Allergic Rhinitis at JoVE.com

acquisition and pre-processing of nasal mucosal samples from rats with allergic rhinitis

Imunofluorescência Multicolorida do Tecido Nasal em Modelo de Rato com Rinite Alérgica

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.

Autor em destaque: Avançando na pesquisa de rinite alérgica com imunofluorescência multicolorida

Marcação por Imunofluorescência em Cortes de Tecido da Mucosa Nasal de Ratos com Rinite Alérgica via Imunoensaio Multicolorido

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.

Watch this Scientific Journal Video about Acquisition and Pre-Processing of Nasal Mucosal Samples from Rats with Allergic Rhinitis at JoVE.com

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

Aquisição e Pré-Processamento de Amostras de Mucosa Nasal de Ratos com Rinite Alérgica

Para começar, com um bisturi, faça uma incisão precisa ao longo dos dois cantos da boca de um rato eutanasiado para expor o tecido muscular. Use uma tesoura de tecido para cortar as articulações do osso zigomático e a mandíbula em ambos os lados do crânio. Em seguida, remova a mandíbula.Use tesoura de tecido para cortar a conexão óssea entre a cavidade nasal e a maxila. Em seguida, use um hemostático para separar a pele da maxila para expor a cavidade nasal. Separar a conexão entre a cavidade nasal e os ossos orbitários de ambos os lados no forame infraorbital e remover a cavidade nasal.Colocar a cavidade nasal extraída em solução de Paraformaldeído a 4% para fixação. Para descalcificar o tecido, lave o tecido fixado com água destilada. Colocar os tecidos numa solução de descalcificação com um volume 20 a 30 vezes superior ao dos tecidos à temperatura ambiente.Quando a descalcificação estiver completa, coloque o tecido em uma caixa de desidratação. Transfira a caixa de desidratação para um desidratador e desidrate o tecido aumentando as concentrações de álcool em diferentes tempos de incubação. Em seguida, colocar o tecido em etanol anidro por 30 minutos por dois ciclos.Em seguida, transfira o tecido para xileno por cinco a 10 minutos e repita, depois mergulhe o tecido em cera por uma hora. Coloque o bloco de cera em de incorporação. Retire os blocos de cera depois de solidificados e apague o bloco de cera.Insira o bloco de cera aparado em um micrótomo de parafina e corte-o em fatias de três micrômetros de espessura. A coloração hematoxilina e eosina dos ratos controle mostrou cílios bem arranjados. A mucosa do septo nasal estava lesada e descolada no grupo da doença, com infiltração neutrofílica notável.

Research

JoVE Journal

Medicine

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

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

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