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.
