Anti anxa1

IHC was performed on 4 μm sections of paraffin-embedded tissues previously prepared. The prepared paraffin sections were dewaxed in fresh xylene and hydrated in gradient alcohol. Antigen repair was carried out with citrate buffer at 100°C for 30 min. Anti-ANXA1 (Cell Signaling Technology, Beverly, MA) was diluted 1:200 and the sections were incubated with the antibody at 4°C overnight. The next day, sections were washed three times on a shaker in PBS, Diaminobenzidine (DAB) reagents (ZSGB-BIO, China) were applied to detect the signal from the antigen-antibody reaction for 5 min. All sections were stained with hematoxylin for 60 s and washed with tap water for 10 min. Finally, the slides were dehydrated in gradient xylene and gradient alcohol, respectively, and were covered with a coverslip. Normal mucous tissue was selected as positive control. Phosphate buffer (PBS) was used to replace the primary antibody and incubated with sections as negative control. All sections were scored by two independent pathologists who were blinded to the clinicopathological information. Depending on the area and intensity of staining, the expression of ANXA1 was scored as either negative (no staining or only weak staining, ≤50% of tumor cells) or positive (moderate staining or strong staining, >50% of tumor cells).