multicolor immunostaining of nasal mucosal tissues of rats with allergic rhinitis

过敏性鼻炎大鼠模型中鼻组织的多色免疫荧光

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.

Watch this Scientific Journal Video about Multicolor Immunostaining of Nasal Mucosal Tissues of Rats with Allergic Rhinitis at JoVE.com

Multicolor Immunostaining of Nasal Mucosal Tissues of Rats with Allergic Rhinitis  

过敏性鼻炎大鼠鼻粘膜组织的多色免疫染色

首先，将苏木精和伊红染色的大鼠鼻粘膜组织切片置于显微镜载物台上。调整显微镜的焦距，直到图像清晰可见。以 40X 的分辨率捕获组织图像。加热柠檬酸盐磷酸盐缓冲液直至沸腾。然后将载玻片从火上移开后将其添加到容器中。加热八分钟直至沸腾后，关火八分钟，然后改用中低火七分钟。让容器自然冷却后，将切片转移到 PBS。使用脱色摇床摇晃切片五分钟。接下来用免疫组织化学笔在干燥的组织周围画一个圆圈。孵育前向切片中加入 3% 过氧化氢溶液。然后像以前一样将切片转移到 PBS 进行洗涤。要阻止，请在标记的圆圈内涂抹 5% 山羊血清。孵育后，小心地除去封闭溶液，并向组织切片中加入一至两毫升FOXP3。然后将切片放入潮湿的腔室中过夜孵育。在PBS中清洗切片后，用滤纸轻轻擦干切片。然后在切片中加入一到两毫升的二抗溶液。接下来，将切片在100微升酪胺信号放大工作溶液中在室温下在黑暗中孵育10分钟。孵育后，用PBS洗涤切片三次，每次五分钟。现在在孵育前将一到两毫升的DAPI染色溶液涂在切片上。在10分钟的PBS洗涤之前，通过在淬灭剂中孵育切片5分钟来淬灭自动荧光。然后用抗荧光淬灭剂密封它们。在 20 倍和 40 倍放大倍率下捕获染色切片的显微图像。对照大鼠的苏木精和伊红染色显示排列良好的上皮细胞和纤毛。该病组的鼻中隔黏膜受损并脱离，伴有明显的中性粒细胞浸润。多免疫荧光染色显示，该病组RORγ T和TCAM1的表达均升高。然而，FOXP3 表达不足。

multicolor-immunostaining-nasal-mucosal-tissues-rats-with-allergic

Research

JoVE Journal

Medicine

Multicolor Immunostaining of Nasal Mucosal Tissues of Rats with Allergic Rhinitis

95 次观看.  Hospital of Chengdu University of Traditional Chinese Medicine. 首先，将苏木精和伊红染色的大鼠鼻粘膜组织切片置于显微镜载物台上。调整显微镜的焦距，直到图像清晰可见。以 40X 的分辨率捕获组织图像。加热柠檬酸盐磷酸盐缓冲液直至沸腾。然后将载玻片从火上移开后将其添加到容器中。加热八分钟直至沸腾后，关火八分钟，然后改用中低火七分钟。让容器自然冷却后，将切片转移到 PBS。使用脱色摇床摇晃切片五分钟。...

视频: Multicolor Immunostaining of Nasal Mucosal Tissues of 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), ...

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

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.