Ab237722

The tissue samples were fixed in 10% formalin and embedded in paraffin. Serial sections (5 µm) were dewaxed, deparaffinized in xylene, and rehydrated with a graded alcohol series. Antigen retrieval in citrate buffer (pH 6.0) was performed with a microwave oven for 20 min. The sections were incubated in 5% goat serum (60 min) to prevent nonspecific antigen binding. Endogenous peroxidase activity was inhibited with 0.3% hydrogen peroxidase for 30 min. Then the primary antibodies were applied and incubated overnight at 4°C. The primary antibodies were diluted as follows: PD‐1 (ab117420, 1:50; Abcam, Cambridge, MA), CD4 (ab237722, 1:4000; Abcam), Foxp3 (ab215206, 1:250; Abcam), and CD8 (bs‐4791R, 1:100; Bioss, Woburn, MA). Then the slides were incubated with a secondary antibody for 60 min. The slides were washed and developed with 3,3‐diaminobenzidine for 10 min. The slides were counterstained with Mayer's hematoxylin and dehydrated with a graded alcohol series.
The evaluation of each slide was performed by a pathologist in a blinded manner. The expression of PD‐1, CD4, and Foxp3 in Peyer's patches was evaluated. The PD‐1‐positive rate (the number of PD‐1‐positive cells / the number of mononuclear cells) was recorded in a ×400 high‐power field.⁹ (link) The number of CD4‐ and Foxp3‐positive cells at five points in the ×400 high‐power field was recorded.¹⁰ (link)