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.

acquisition-pre-processing-nasal-mucosal-samples-from-rats-with

Research

JoVE Journal

Medicine

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

262 visualizações.  Hospital of Chengdu University of Traditional Chinese Medicine. 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 conex...

Vídeo: 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), 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.

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

Medicina

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.

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.