Pd 1 antibody

All the specimens were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 24 h and embedded in paraffin and then sectioned into 4-μm sections and mounted on glass slides. After dewaxing, the slides were treated with 3% hydrogen peroxide in methanol and blocked with a biotin blocking kit (Dark, Germany). After blocking, the slides were incubated overnight with PD-1 antibody (1:1000; Proteintech, USA) or PD-L1 (1:1000; Proteintech, USA) or P16 (1:1000; Santa Cruz, USA) in a humid chamber at 4°C, washed three times with phosphate buffered saline (PBS), incubated for 1 h with biotinylated goat anti-mouse antibody, and then stained with 3,3′-diaminobenzidine tetrahydrochloride. Finally, these sections were stained with hematoxylin and observed under a microscope. Semiquantitative immunohistochemistry (IHC) was used to detect the protein expression levels of epidermal fatty acid binding protein, according to the following standard scores: "0" (negative staining), "1" (weak staining), "2" (moderate staining), and "3" (strong staining). The final score was calculated as the percentage of positive expression multiplied by the intensity score, which was independently determined by two pathologists. The median IHC score was used as the cut-off to define high and low expression levels.