2.8 | Immunohistochemical staining
We collected nasal mucosal specimens from patients who met the diagnostic criteria described previously. During surgery, the nasal mucous was removed, fixed in 4% paraformaldehyde for 48 h, and then embedded in paraffin. The antigens were extracted and incubated overnight with ZO-1 antibody (Proteintech, China) and Col-a1 antibody (CST, USA) at 4 °C. On the next day, the slices were washed with PBS, followed by incubation with secondary antibodies at room temperature for 1 h. Microscopically, positive expression of Col-a1 and ZO-1 was confirmed if the particles turned brownish yellow or tan. The positive field of vision under 400× magnification was considered from each stained section. After image collection, we used ImageJ software to analyse the total area and cumulative optical density of the positive cells. The average optical density (AOD) was used for statistical analysis.