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

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 treatments of 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 5 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 epithelial cells and celia. The nasal septum mucosa was damaged and detached in the disease group, with notable neutrophil infiltration. 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 autofluorescence by incubating the slices in the quencher for 5 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 celia. 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 TICAM-1 were increased in the disease group.

However, Foxp3 was under expressed.