Immunohistochemical Analysis of COX-2 Expression

Briefly, formalin-fixed paraffin-embedded tissue sections were deparaffinized using xylene, rehydrated, and washed in phosphate-buffered saline (pH 7.0), and the remaining protocols were followed as per the method described earlier [26 (link),27 (link)]. The COX-2 protein of Abcam, Cambridge, U.K. was used as primary antibodies and incubated for 1 h at 4 °C, followed by incubation with the secondary antibody for 60 min, then streptavidin–biotin enzyme complex for 1 h. Diaminobenzidine (DAB) (Abcam, Cambridge, UK, ab 64259) chromogen was then applied accordingly, and hematoxylin was used as a counterstain. Finally, the results were analyzed under a light microscope.