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.